Intrinsic oxidase-mimicking activity of nitrite upon visible light illumination and its colorimetric detection in saliva.

Small molecules with enzyme-like properties have recently attracted considerable attention. Herein, we discovered that nitrite possesses intrinsic oxidase-mimicking activity upon visible light, catalyzing the oxidation of the typical chromogenic substrate in the absence of H2O2. Notably, nitrite exhibited a markedly high value of Kcat, approximately 4, 7, and 4000-fold greater than that of Acr+-Mes, Eosin Y, and Diacetyl, respectively. Comprehensive investigation elucidated that O2•⁻ and •OH are the primary reactive oxygen species (ROS) responsible for the oxidation of 3,3',5,5'-tetramethylbenzidine dihydrochloride hydrate (TMB). Leveraging the linear correlation between the absorbance of oxidized TMB (oxTMB) at 652 nm and nitrite concentration, a simple colorimetric approach for nitrite detection was successfully established in the range of 1-75 µM with a detection limit of 0.14 µM. Moreover, the proposed strategy could be applied to determine the nitrite concentration in saliva, exhibiting a great prospect for clinical diagnosis. This work contributes novel insights into the exploration of small-molecule enzyme mimics.

Detecting uric acid base on the dual inner filter effect using BSA@Au nanoclusters as both peroxidase mimics and fluorescent reporters.

Fluorescent bovine serum albumin-protected gold nanoclusters (BSA@Au NCs) can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to produce blue oxTMB for its peroxidase-like activity. The two absorption peaks of oxTMB overlapped with the excitation and emission peaks of BSA@Au NCs, respectively, causing efficient quenching on the fluorescence of BSA@Au NCs. The quenching mechanism can be attributed to the dual inner filter effect (IFE). Based on the dual IFE, BSA@Au NCs were utilized as both peroxidase mimics and fluorescent reporters for H2O2 detection and further for uric acid detection with uricase. Under optimal detection conditions, the method can be used to detect H2O2 ranging 0.50-50 µM with a detection limit of 0.44 µM and UA ranging 0.50-50 µM with a detection limit of 0.39 µM. The established method had been successfully utilized for the determination of UA in human urine, with massive potential in biomedical applications.

Colorimetric detection of biothiols and Hg2+ based on the peroxidase-like activity of GTP.

Guanosine-5'-triphosphate (GTP) not only plays a key role in a majority of cellular processes but also be proposed as a peroxidase-like mimic. Compared with nanozymes, GTP shows good tolerance under harsh conditions, which can be used to construct an easy colorimetric analysis for the detection of biomolecules. Here, on the basis of the peroxidase-like activity of GTP which can catalyze the oxidation of 3,3',5,5'-tetramethyl benzidine dihydrochloride (TMB), colorimetric sensing was established for biothiols and Hg2+. Biothiols reduced the oxTMB back to colorless TMB, and Hg2+ restored the formation of oxTMB, leading to the recovery of color. This method not only provides a platform for the detection of metal ions and biothiols, but also shows that GTP has great potential for analytical detection.

Ru(bpy)32+ as a photoinduced oxidase mimic for colorimetric detection of biothiols.

We have found that tris (2,2'-bipyridyl) ruthenium (II) (Ru(bpy)32+) possesses a high photo-induced oxidase-like activity and is capable of catalyzing the color reaction of 3,3',5,5'-tetramethylbenzidine (TMB) with dissolved oxygen. Ru(bpy)32+ has a catalytic constant (Kcat) that is twice as high as that of fluorescein, 170 and 275-fold higher than that of 9-mesityl-10-methyl acridine and Eosin Y, respectively. Electron spin resonance spectroscopy (ESR) and radical scavenging experiments have verified the major active radicals involved in the color reaction are •OH. A colorimetric biothiol assay has been successfully developed for the oxidase-like activity of Ru(bpy)32+ can be suppressed by sulfhydryl compounds. A linear dependence between the decrease in absorbance and the logarithm of thiol concentrations can be found ranging from 5.0 to 50 µM, with a detection limit of 1.0 µM. This work reveals a new oxidase mimic with high catalytic activity and will facilitate the utilization of this oxidase mimic in biochemical analysis.

Colorimetric detection of ATP by inhibiting the Peroxidase-like activity of carbon dots.

Adenosine triphosphate (ATP) is the main energy currency for cells and an important biomolecule involved in cellular reactions, whose abnormal levels are closely related to physical disease, thus it is extremely important to establish a convenient, fast and simple ATP monitoring method. Toward this end, we developed a facile method for colorimetric detection of ATP on the basis of the inhibiting effect of ATP on the peroxidase-like activity of carbon dots (CDs). The detection principle of this method was utilizing the peroxidase-like activity of CDs, which catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 to generate blue products. However, the introduction of ATP in the system can inhibit the generation of blue products, so ATP can be colorimetric detected. This method exhibited high sensitivity with a detection limit of 34 nM and a wide linear range (0.050-2.0 µM). The as-proposed colorimetric ATP sensor was capable of detecting ATP in real samples accurately.

Metal-free colorimetric detection of pyrophosphate ions by the peroxidase-like activity of ATP.

Pyrophosphate (P2O74-, PPi) plays a vital role in ecological environment. Its elevated levels in water bodies can lead to eutrophication. Hence, its detection is extremely significant. Whereas most of the existing methods for the actual detection of PPi may cause environmental pollution or suffer from operational complexity. In this study, we introduced a sensitive and selective method for detecting PPi based on the fact that PPi can inhibit the peroxidase-like activity of adenosine 5'-triphosphate (ATP). This strategy not only eliminated the complexity of material preparation (ATP is commercialized), but also addressed the general need for metal ions in detecting PPi. The dynamic range of PPi detection was 1.0-200 µM and the detection limit was 74 nM. In addition, this strategy had been successfully applied to the determination of PPi in tap water and lake water. This work extends the application of natural biological small molecule ATP in the analysis and provides an innovative thought for the metal-free detection of PPi.

Enhancing the peroxidase activity and decreasing the protease activity of ficin with rational modification and its application to one-step colorimetric detection of glucose.

Ficin has dual enzyme activity, i.e., protease and peroxidase-like activity. In some respects, its application is limited by the protease activity of ficin. Herein, we used tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to break the three pairs of disulfide bonds of ficin, and then blocked the free thiol groups with N-ethylmaleimide (NEM) to synthesize ficin-TN. The results showed that ficin-TN had increased peroxidase-like activity and reduced protease activity. According to this phenomenon, we have exploited a colorimetric method with high sensitivity and selectivity for the one-step detection of glucose. Comparing with ficin, ficin-TN has wider detection range (0.1-300 µM) and lower detection limit (88 nM), and our method is simpler and more timesaving than other two-step methods. Furthermore, the actual appliances of ficin-TN for glucose detection in human serum have been illustrated with satisfied result, suggesting that its promising utilization in various fields.

Reducing background absorbance via a double-lock strategy for detection of alkaline phosphatase and α-fetoprotein.

Lowering the background signal for more sensitive analysis of determinands is as important as amplifying the target signal. The photoinduced oxidase of fluorescein has been reported, which can catalyze the oxidization of common substrates in a few minutes. As a metaphor for locks and keys, we designed double locks confining the activity of fluorescein to reduce the background absorbance during colorimetric detection. The first lock inhibits the main activity of fluorescein by phosphating. The second lock almost completely deactivates fluorescein by forming coordination nanoparticles (CNPs) via the self-assembly of cerium chloride and fluorescein diphosphate (FDP). The Ce-FDP CNPs are characterized by scanning electron microscope (SEM), dynamic light scattering (DLS), Fourier transform infrared spectrometer (FTIR), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and energy dispersive spectrum (EDS), which show electrostatic formation and amorphous character in the morphology. Alkaline phosphatase (ALP), the key to release fluorescein, can destroy Ce-FDP CNPs along with decomposing FDP by degrading phosphate groups. Therefore, a novel colorimetric strategy for sensitive detection of ALP is established. The detection of α-fetoprotein (AFP) is further succeeded by labeling AFP antibody with ALP. By dramatically reducing the background absorbance, the detection limits of ALP and AFP are as low as 0.014 mU/mL and 0.023 ng/mL, respectively. This convenient, brief, sensitive assay provides a promising prospect for clinical diagnosis. Graphical abstract.

Bifunctional antibody and copper-based metal-organic framework nanocomposites for colorimetric α-fetoprotein sensing.

Cu2+ are found to greatly reduce the photoinduced oxidase activity of fluorescein and then inhibit the chromogenic reaction catalyzed by fluorescein. A simple colorimetric assay for Cu2+ is established. Based on this, bifunctional nanocomposites of α-fetoprotein (AFP) antibody (Ab) and copper-based metal-organic framework (Ab2@Cu-MOF) are synthesized by the simple self-assembly of AFP Ab2, Cu2+, and 4,4'-dipyridyl: the binding site of AFP Ab2 exposed on the surface of the nanocomposites can specifically recognize AFP antigen; Cu2+ in nanocomposites can inhibit the visible light-induced activity of fluorescein. The structure of Ab2@Cu-MOF disintegrate and Cu2+ is released in an acetate buffer solution. The higher the amount of AFP antigens, the more significant the inhibitory effect. Thus, the Ab2@Cu-MOF immunoassay for AFP determination is established using 3,3',5,5'-tetramethylbenzidine as chromogenic substrate with a detection limit of 35 pg.mL-1. This simple, cheap, and sensitive method sheds substantial light on practical clinical diagnosis. Meanwhile, the mechanism of inhibition is revealed to facilitate the targeted selection of enzyme regulators. Graphical abstract Diagrammatic illustration of Cu2+ detection (part a) and Ab2@Cu-MOF immunoassay for sensing α-fetoprotein based on the synthesized Ab2@Cu-MOF nanocomposites (parts a and b).

A signal amplification strategy for prostate specific antigen detection via releasing oxidase-mimics from coordination nanoparticles by alkaline phosphatase.

A novel signal amplification method for prostate specific antigen (PSA) is developed by freeing fluorescein with photoinduced oxidase-like activity from coordination nanoparticles (CNPs) in the presence of alkaline phosphatase (ALP). CNPs loaded with fluorescein (F@CNPs) are obtained in aqueous solution by self-assembly using Tb3+ as metal ion, guanosine monophosphate (5'-GMP) as ligand, and fluorescein as signal molecule. The F@CNPs display outstanding properties of simple synthesis, low cost, good water solubility, negligible leakage and satisfactory load capacity. Fluorescein is quantitatively encapsulated in CNPs with a binding ratio of 92.72%. Meanwhile, ALP can specifically hydrolyze the phosphate group of 5'-GMP ligand, triggering the destruction of F@CNPs and leakage of fluorescein. Fluorescein, a photoinduced oxidase mimic, can catalyze the oxidation of non-fluorescent Amplex UltraRed (AUR) into fluorescent resorufin under LED lamp. This strategy exhibits good sensitivity for ALP detection. In addition, a new immunoassay for PSA is validated by labelling ALP on PSA antibody. The low detection limit of 0.04 ng mL-1 in detecting PSA is appropriate for PSA detection in real samples. Therefore, the work not only establishes a new strategy for ALP and PSA determination, but also provides a new conception for putting photoinduced oxidase-like fluorescein in practical application.

Ficin encapsulated in mesoporous metal-organic frameworks with enhanced peroxidase-like activity and colorimetric detection of glucose.

Ficin has been reported to possess peroxidase activity, but its applications in some respects have been limited because of its relatively low activity. Herein, a mesoporous metal-organic framework, PCN-333(Fe), was synthesized, which was selected to encapsulate ficin to form ficin@PCN-333(Fe). Compared with ficin, the peroxidase-like activity of ficin@PCN-333(Fe) toward 3,3',5,5'-tetramethylbenzidine (TMB) oxidation was about 3 times increase in the presence of H2O2, and followed classical Michaelis-Menten model. The kinetic parameters showed that stronger affinity and higher catalytic constant (Kcat) of ficin@PCN-333(Fe) to both TMB and H2O2 compared with ficin, and Kcat of ficin@PCN-333(Fe) was increased by 3.65 folds and 3.59 folds for TMB and H2O2, respectively. Taking advantages of higher catalytic property of ficin@PCN-333(Fe), we developed a colorimetric method with high sensitivity and selectivity to detect glucose, which displayed a good linear response toward glucose in the range of 0.5-180 µM with a limit of detection of 97 nM. Furthermore, ficin@PCN-333(Fe) has been proven to successfully detect glucose in human serum, implying its great potentialities and wide applications as peroxidase mimics.

A siramesine-loaded metal organic framework nanoplatform for overcoming multidrug resistance with efficient cancer cell targeting.

Cancer is the leading cause of death and the most important obstacle to increasing life expectancy. With the sophisticated design and research of anticancer drugs, multidrug resistance to chemotherapy has become more and more common. After the emergence of multidrug resistance, the development of a new drug is beset with difficulties. The repurposing of non-anticancer drugs is thus a timely strategy for cancer therapy. Here, we highlight the potential of repurposing siramesine, a central nervous system drug for antitumor research and we construct a metal organic framework-based nanoplatform for effective intracellular accumulation and pH-response siramesine release. The released drug induces lysosome membrane permeabilization, leading to lysosomal cathepsins leakage and then results in cell apoptosis. Due to the modification of folic acids, the constructed drug delivery nanosystem shows good biocompatibility and efficient cancer cell targeting. Importantly, the drug delivery system shows enhanced anticancer efficacy in vitro, which not only effectively kills cancer cells but also kills multidrug resistant cells. Thus, the drug delivery nanosystem constructed in this study is thought to become a promising anticancer agent for cancer therapy and even overcoming multidrug resistance, which provides good prospects for biomedical applications.

Cetyltrimethylammonium chloride-loaded mesoporous silica nanoparticles as a mitochondrion-targeting agent for tumor therapy.

Mitochondria play an important role in supplying cellular energy, cell signaling and governing cell death. In addition, mitochondria have also been proved to be essential for tumor generation and development. Thus, mitochondrion-targeting therapeutics and treatments have emerged as promising strategies against cancer. However, the lack of mitochondrion-targeting agents has limited their application. To this end, we report cetyltrimethylammonium chloride-loaded mesoporous silica nanoparticles conjugated with human serum albumin (CTAC@MSNs-HSA) as a mitochondrion-targeting agent for anticancer treatment. As the structure-directing agent in the synthesis of MSNs, CTAC is stored within MSNs. Due to their desirable size and HSA receptor-mediated transcytosis, CTAC@MSNs-HSA show great cellular uptake and enhanced accumulation in the cytoplasm. Positively charged CTAC could actively target mitochondria by interacting with the negatively charged mitochondria membrane, and then lead to the dysfunction of mitochondria by decreasing mitochondrial potential and intracellular ATP levels, resulting in the necrosis and apoptosis of MCF-7 cells. Therefore, significant antitumor activity is observed by in vitro studies. Moreover, in vivo studies confirm that the CTAC@MSNs-HSA are able to induce cancer cell death and efficiently inhibit tumor growth. These results demonstrate the potential of CTAC@MSNs-HSA in cancer therapeutics as well as providing insights into mitochondrion-targeting treatment.

Enhancing the peroxidase-like activity of ficin by rational blocking thiol groups for colorimetric detection of biothiols.

The peroxidase-like activity of ficin is relatively low, which limits its application. It was found that thiol groups of ficin could inhibit its peroxidase-like activity. So, two procedures, i.e., direct blocking with N-ethylmaleimide (NEM), or using tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to interrupt disulfide bonds then blocking thiol groups with NEM, were applied to block thiol groups of ficin, ficin-NEM (ficin-N) and ficin-TCEP-NEM (ficin-TN) were produced, respectively. The blocking of thiol groups accelerated the peroxidase activity dramatically. The peroxidase catalytic activity of ficin-N and ficin-TN toward the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) oxidation by H2O2 was about 2.5-fold and 5-fold increase compared with ficin, respectively, which accompanied a color change from colorless to blue and followed classic Michaelis-Menten model. The kinetic parameters indicated that higher affinity of ficin-N (Km = 0.31) and ficin-TN (Km = 0.39) to H2O2 compared with ficin (Km = 0.58), and ficin-TN had the highest Kcat which increased by 6.5 times and 4.5 times for TMB and H2O2, respectively. According to these findings, a colorimetric method with high sensitivity for the detection of biothiols was developed due to sulfhydryl compounds inhibited the peroxidase activity of ficin. Comparing with ficin and ficin-N, ficin-TN had the widest detection range (0.01-16 µM) and the lowest detection limit (3 nM). The practical applications of ficin-TN for biothiol determination in human serum samples have been demonstrated with satisfactory results. Ficin-N and ficin-TN are promising to apply to the bioanalysis.

Tuning the fluorescence performance of carbon dots with a reduction pathway.

A reduction pathway was developed to tune the fluorescence quantum yield (QY) and emission wavelength of carbon dots (CDs). Reducing the original CDs (QY ca. 1.55%, maximum emission ca. 520 nm) with NaBH4 increased QY to 7.25% and blue-shifted the maximum emission to 450 nm. Treating the original CDs with LiAlH4 increased QY to ca. 7.44% and blue-shifted the maximum emission to 345 nm. The distinct fluorescence characteristics of the original CDs, NaBH4-treated CDs, and LiAlH4-treated CDs were conferred by their differing surface groups. Moreover, the original CDs, NaBH4-treated CDs, and LiAlH4-treated CDs responded differently to a specific oxidizer of hydroxyl groups. The increase in the fluorescence QY and blue-shift of the emission wavelength were explained. This study provides a foundation for the preparation and tuning of CDs with excellent fluorescence performance, as well as for the development CD-based fluorescent probes.

One-pot synthesis of a composite consisting of the enzyme ficin and a zinc(II)-2-methylimidazole metal organic framework with enhanced peroxidase activity for colorimetric detection for glucose.

An enzyme-metal organic framework (MOF) composite with saucer-like structure was prepared via one-pot synthesis from ficin (a cysteine proteolytic enzyme with POx activity), zinc(II) ions and 2-methylimidazole. The composites exhibit a 2.5-fold higher catalytic activity and stronger affinity for substrates compared to free ficin. This was exploited to design a colorimetric assay for the determination of glucose. The addition of glucose oxidase causes the formation of H2O2 which is catalytically oxidized by ficin to form a blue coloration that can be measured at 652 nm. The assay has a 0.12 µM detection limit and excellent selectivity. It was successfully applied to the determination of glucose in diluted serum samples. Graphical abstract Schematic presentation of the synthesis process and the enhanced peroxidase activity of ficin@MOF composites. Ficin was immobilized in a new saucer-like shape of Ficin@MOF composites, which showed higher peroxidase activity than free ficin. Scheme and graphical abstract contains poor quality of text.We have attach a new picture with 600 dpi as scheme and graphical abstract.

Competitive method for fluorescent dopamine detection in cerebrospinal fluid based on the peroxidase-like activity of ficin.

Dopamine (DA), a catecholamine neurotransmitter, is considered to be an important indicator for mental diseases detection in the clinic. In this study, a novel fluorescent sensing platform consisting of the ficin-H2O2-tyramine system for determining DA in cerebrospinal fluids (CSF) was established. The proposed method is based on the fact that ficin, a mimetic peroxidase, can catalyze H2O2 decomposition into OH radicals, which can oxidize non-fluorescent tyramine into fluorescent dityramine. When DA was introduced, DA can compete with tyramine for OH and resulting in the oxidation reaction of tyramine inhibited along with the fluorescence intensity of the system decreased, which provides a unique strategy for fluorescence detection of DA. Under optimal conditions, the fluorescence intensity decreased linearly with the DA level over a wide concentration range from 0.05 to 12.0 µM (R2 = 0.995) with a detection limit of 46 nM (3σ/k). More importantly, the proposed sensing approach exhibits high sensitivity, good selectivity and has been successfully applied to DA sensing in complex biological samples, which made it hold great potential for DA determination in chemical and biological analytical applications.

Quantum dots-based sandwich immunoassay for sensitive detection of Alzheimer's disease-related Aß1-42.

Amyloid-beta peptide 1-42 (Aß1-42) is known as a component of amyloid plaques in association with Alzheimer's disease. Herein, we developed a reliable and remarkably sensitive sandwich immunoassay to detect the Aß1-42 using quantum dots (QDs) as fluorescent label. In the presence of Aß1-42, the biotinylated Anti-beta Amyloid 1-16 (N-Ab) recognized the target and formed C-Ab-Aß1-42-N-Ab sandwich immunocomplexes. Then Streptavidin-QDs conjugated to biotinylated N-Ab and the concentration of Aß1-42 was determined by detecting the fluorescence intensity in the supernatant. This method is faster and more efficient than the previous approach we reported. It also has reasonable sensitivity and selectivity. Under the optimized conditions, the linear range is 5.0 to 100 pM (0.023-0.45 ng/mL) and the detection limit is 1.7 pM (7.6 pg/ mL). In addition, this method has been successfully applied to detect the Aß1-42 in human cerebrospinal fluid sample.

Enhancing the peroxidase-like activity of ficin via heme binding and colorimetric detection for uric acid.

Ficin, a classical sulfhydryl protease, was found to possess intrinsic peroxidase-like activity. In this paper, we have put forward a novel strategy to improving the peroxidase-like activity of ficin through binding heme. Heme-ficin complexes were successfully obtained by simple one-step syntheticism. The results demonstrated that the catalytic activity and efficiency of heme-ficin complexes were about 1.7 times and 3 times higher than those of native ficin, respectively. Taking advantages of the high peroxidase-like activity, the heme-ficin complexes were used for colorimetric determination of uric acid with a low detection limit of 0.25 µM. Based on the excellent selectivity and sensitivity, we detected the concentration of uric acid in human serum successfully. On the basis of these findings, the heme-ficin complexes are promising for wide applications in various fields. Thus we not only optimized the peroxidase-like activity of the ficin, but also established a new strategy for development of artificial enzyme mimics by mimicking the architecture of the active site in horseradish peroxidase.

Acidic amino acids: A new-type of enzyme mimics with application to biosensing and evaluating of antioxidant behaviour.

Nanomaterials have triggered tremendous interest to mimick peroxidase but rarely attention has been paid to small molecules. Herein we first found that acidic amino acids including l-glutamic acid (L-Glu) and l-aspartic acid (L-Asp) exhibited an intrinsic peroxidase-like activity, endowing acidic amino acids with the capability of catalysing the oxidation of the peroxidase substrates 3,3',5,5'-tetramethylbenzidine (TMB) to produce color reaction in the presence of H2O2. Reaction mechanism was further investigated by means of electron spin resonance spectroscopy (ESR), enzyme kinetics assay and quantum theoretical calculations, to verify and provide a good deal of insight into the catalytic process. Based on the above discovery, a colorimetric platform was successfully developed for sensing glucose in the range of 0.10 µM to 10 µM with a detection limit of 40 nM, as well as evaluating the inhibitory effect of antioxidants on reactive oxygen species. This extraordinary finding not only extends the new biological function of acidic amino acids, but also opens new opportunities to deepen the knowledge of the new class of small molecule enzymes.