Conventional Colorimetric Assay Research Articles

Naturally occurring quercetin and myricetin as potent inhibitors for human ectonucleotide pyrophosphatase/phosphodiesterase 1

Ecto-nucleotide pyrophosphatases/phosphodiesterases 1 (ENPP1) is a key enzyme in purinergic signaling pathways responsible for cell-to-cell communications and regulation of several fundamental pathophysiological processes. In this study, Kyoto Green, a rapid chemical sensor of pyrophosphate, was employed to screen for effective ENPP1 inhibitors among five representative flavonoids (quercetin, myricetin, morin, kaempferol, and quercetin-3-glucoside), five nucleosides (adenosine, guanosine, inosine, uridine, and cytidine), and five deoxynucleosides (2′- and 3′-deoxyadenosine, 2′-deoxyguanosine, 2′-deoxyinosine, and 2′-deoxyuridine). Conventional colorimetric, fluorescence, and bioluminescence assays revealed that ENPP1 was effectively inhibited by quercetin (Ki ~ 4 nM) and myricetin (Ki ~ 32 nM) when ATP was used as a substrate at pH 7.4. In silico analysis indicated that the presence of a chromone scaffold, particularly one containing a hydroxyl group at the 3′ position on the B ring, may promote binding to the active site pocket of ENPP1 and enhance inhibition. This study demonstrated that the naturally derived quercetin and myricetin could effectively inhibit ENPP1 enzymatic activity and may offer health benefits in arthritis management.

Fluorometric assay for phenol oxidase activity in soils and its controlling variables

Phenolic compounds constitute the major component of recalcitrant carbon pools in soils and undergo degradation by enzymes such as phenol oxidases. Despite their significance, a common, appropriate assay to determine its activity is yet to be found. Until recently, only colorimetric assays, using substrates such as L-DOPA, pyrogallol, and ABTS, have been dominantly employed to measure phenol oxidase activity, despite their drawbacks, such as interference of soil background color, unavailability of a commercial product, and low sensitivity. Therefore, this study aimed to identify the optimal conditions for the novel fluorometric Amplex Red assay, compare the effectiveness and reproducibility of this assay with the conventional colorimetric L-DOPA assay, and reveal the key controlling variables of phenol oxidase activity by applying it to various soil types. Results showed that the optimal phenol oxidase activity can be determined at a substrate concentration of 500 μM and when the soil suspension is prepared with sonication only, using deionized water as a solvent. The comparison of the Amplex Red and L-DOPA assay indicated that the fluorometric assay has greater reproducibility, with a significantly smaller coefficient of variation (19.9 % and 107 %, respectively). For the L-DOPA assay, there was substantial variability between replicates and strong dependence on soil suspension preparation methods. Although this study reveals that the Amplex Red assay is more suitable for measuring phenol oxidase activity, additional research on other soil types is important to confirm its applicability, and adjustments to incubation conditions may be necessary for specific soil types. The application of the Amplex Red assay on various soil types uncovered soil pH and organic matter as the two most important factors controlling phenol oxidase activity, which aligned with previous findings. A positive linear correlation between phenol oxidase activity and soil pH (R = 0.734, P ≤ 0.000) but a negative exponential correlation with soil organic matter content (R = −0.547, P ≤ 0.01).

Gold nanostructure-integrated conductive microwell arrays for uniform cancer spheroid formation and electrochemical drug screening

Cancer spheroids, which mimic distinct cell-to-cell and cell-extracellular matrix interactions of solid tumors in vitro, have emerged as a promising tumor model for drug screening. However, owing to the unique characteristics of spheroids composed of three-dimensionally densely-packed cells, the precise characterizations of cell viability and function with conventional colorimetric assays are challenging. Herein, we report gold nanostructure-integrated conductive microwell arrays (GONIMA) that enable both highly efficient uniform cancer spheroid formation and precise electrochemical detection of cell viability. A nanostructured gold on indium tin oxide (ITO) substrate facilitated the initial cell aggregation and further 3D cell growth, while the non-cytophilic polymer microwell arrays restricted the size and shape of the spheroids. As a result, approximately 150 human glioblastoma spheroids were formed on a chip area of 1.13 cm2 with an average diameter of 224 μm and a size variation of only 5% (±11.36 μm). The high uniformity of cancer spheroids contributed to the stability of electrical signals measuring cell viability. Using the fabricated GONIMA, the effects of a representative chemotherapeutic agent, hydroxyurea, on the glioblastoma spheroids were precisely monitored under conditions of varying drug concentrations (0–0.3 mg/mL) and incubation times (24–48 h). Therefore, we conclude that the newly developed platform is highly useful for rapid and precise in vitro drug screening, as well as for the pharmacokinetic analyses of specific drugs using 3D cellular cancer models.

Magnetic supercluster particles for highly sensitive magnetic biosensing of proteins.

A strategy is reported to improve the detection limits of current giant magnetoresistance (GMR) biosensors by augmenting the effective magnetic moment that the magnetic tags on the biosensors can exert. Magnetic supercluster particles (MSPs), each of which consists of ~ 1000 superparamagnetic cores, are prepared by a wet-chemical technique and are utilized to improve the limit of detection of GMR biosensors down to 17.6 zmol for biotin as a target molecule. This value is more than four orders of magnitude lower than that of the conventional colorimetric assay performed using the same set of reagents except for the signal transducer. The applicability of MSPs in immunoassay is further demonstrated by simultaneously detecting vascular endothelial growth factor (VEGF) and C-reactive protein (CRP) in a duplex assay format. MSPs outperform commercially available magnetic nanoparticles in terms of signal intensity and detection limit.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00604-022-05354-x.

A novel galactose electrochemical biosensor intended for point-of-care measurement of quantitative liver function using galactose single-point test.

Liver disease has emerged as a healthcare burden because of high hospitalization rates attributed both to steatohepatitis and to severe hepatic toxicity associated with changes of drug exposure. Early detection of hepatic insufficiency is critical to preventing long-term liver damage. The galactose single-point test is recommended by the US FDA as a sensitive means to quantify liver function, yet the conventional method used for quantitation of circulating galactose still relies on the standard colorimetric method, requiring time-consuming and labor-intensive processes, and is confined to the medical laboratory, thus limiting prevalence. To facilitate time- and cost-effective disease management particularly during a pandemic, a pocket-sized rapid quantitative device consisting of a biosensor and electrochemical detection has been developed. An in vitro validation study demonstrated that the coefficient of variation was less than 15% and deviations were between -4 and 14% in the range of 100-1500μg/mL. The device presented good linear fit (correlation coefficient, r = 0.9750) over the range of 150-1150µg/mL. Moreover, the device was found to be free from interference of common endogenous and exogenous substances, and deviated hematocrit, enabling a direct measurement of galactose in the whole blood without sample pre-treatment steps. The clinical validation comprising 118 subjects showed high concordance (r = 0.953) between the device and the conventional colorimetric assay. Thus, this novel miniaturized device is reliable and robust for routine assessment of quantitative liver function intended for follow-up of hepatectomy, drug dose adjustment, and screening for galactosemia, allowing timely and cost-effective clinical management of patients.

Action of Varespladib (LY-315920), a Phospholipase A2 Inhibitor, on the Enzymatic, Coagulant and Haemorrhagic Activities of Lachesis muta rhombeata (South-American Bushmaster) Venom.

Varespladib (VPL) was primarily developed to treat inflammatory disturbances associated with high levels of serum phospholipase A2 (PLA2). VPL has also demonstrated to be a potential antivenom support agent to prevent PLA2-dependent effects produced by snake venoms. In this study, we examined the action of VPL on the coagulant, haemorrhagic and enzymatic activities of Lachesis muta rhombeata (South-American bushmaster) venom. Conventional colorimetric enzymatic assays were performed for PLA2, caseinolytic and esterasic activities; in vitro coagulant activities for prothrombin time (PT) and activated partial thromboplastin time (aPTT) were performed in rat citrated plasma through a quick timer coagulometer, whereas the dimensions of haemorrhagic haloes obtained after i.d. injections of venom in Wistar rats were determined using ImageJ software. Venom (1 mg/ml) exhibited accentuated enzymatic activities for proteases and PLA2 in vitro, with VPL abolishing the PLA2 activity from 0.01 mM; VPL did not affect caseinolytic and esterasic activities at any tested concentrations (0.001–1 mM). In rat citrated plasma in vitro, VPL (1 mM) alone efficiently prevented the venom (1 mg/ml)-induced procoagulant disorder associated to extrinsic (PT) pathway, whereas its association with a commercial antivenom successfully prevented changes in both intrinsic (aPTT) and extrinsic (PT) pathways; commercial antivenom by itself failed to avoid the procoagulant disorders by this venom. Venom (0.5 mg/kg)-induced hemorrhagic activity was slightly reduced by VPL (1 mM) alone or combined with antivenom (antivenom:venom ratio 1:3 ‘v/w’) in rats, with antivenom alone producing no protective action on this parameter. In conclusion, VPL does not inhibit other major enzymatic groups of L. m. rhombeata venom, with its high PLA2 antagonize activity efficaciously preventing the venom-induced coagulation disturbances.

Monitoring the mechanism of anti-cancer agents to inhibit colorectal cancer cell proliferation: Enzymatic biosensing of glucose combined with molecular docking

Glucose, a major energy source in cellular metabolism, has a significant role in cell growth. The increase in glucose uptake is a distinguishing hallmark in cancer cells. A key step in glucose utilization is the transport of glucose to the cancer cells for supplying their additional energy. The glucose transporter (or GLUT) family is a membrane protein which facilitates the uptake of glucose in most cancer cell types. Given the increased glucose level in cancer cells and the regulatory role of GLUTs in glucose uptake, it is required to combine both experimental and theoretical studies to develop new methods to monitor cell proliferation. Herein, for the first time, a new strategy was proposed to evaluate the cell proliferation of HT-29 based on glucose consumption in the presence of resveratrol (RSV) as an anticancer agent. A hybrid nanocomposite of carbon nanofibers and nitrogen-doped graphene quantum dots was used to design an enzymatic sensor for the selective and sensitive determination of glucose in cancer cells. The results obtained from the voltammetric technique were compared with the conventional colorimetric assay. A good correlation was observed between the proliferation rate and glucose utilization by cancer cells. As it was observed, RSV induces a decrease in glucose consumption, indicating lower glucose uptake efficiency for HT-29 cells. Molecular docking studies reveal that RSV can block the interaction of glucose with the GLUT family. This is one of the possible mechanisms for the decrease of glucose level followed by the reduction of cell proliferation in the presence of RSV. Compared with traditional methods, in vitro electrochemical techniques benefit from simple, nontoxic, sensitive and low-cost detection assays and hence serve as a novel tool to pursue the growth inhibition of cancer cell in response to anti-cancer agents.

Precise Electrical Detection of Curcumin Cytotoxicity in Human Liver Cancer Cells

Curcumin is a polyphenol extracted from the roots of Curcuma plants that exerts potential anticancer effects. However, owing to its low toxicity, curcumin is known to be effective only at high doses. Here, we report the highly sensitive assessment of curcumin toxicity in human liver cancer cells, which was facilitated using a fabricated conductive platform and an electrochemical detection method. To identify the best platform for assessing cell viability, both electrochemical deposition time (0–150 s) and RGD peptide concentrations (0–0.1 mg/mL) were varied. Unlike other cell types, liver cancer cells showed the highest electrical signals without the modification of cell adhesion peptides. With 120 s of gold deposition under peptide-free conditions, 10,000 cells/chip were detectable within a linear range of 10,000–500,000 cells. Notably, even at low concentrations (e.g., 20 µM), curcumin could inhibit liver cancer cell-specific electrical signals by 45%, which would be undetectable in conventional colorimetric assay (CCK-8). The half-maximal inhibitory concentration (IC50) of curcumin for HepG2 cells was 23.63 µM, which indicates 3.71-fold higher sensitivity than that determined in the CCK-8 assay (IC50 = 87.88 µM). Hence, the developed conductive cell culture platform, in combination with the electrochemical detection method, could serve as a promising tool for detecting toxicity and/or anticancer activity of food-derived compounds in cancer/normal cells.

A whole area scanning-enabled direct-counting strategy for studying blocking efficiency in mitigating protein-solid surface binding.

The study of protein-solid surface binding as well as blocking efficiency of blocking agents plays an important role in the development of high-performance immunoassays. Although conventional colorimetric based assays are widely employed to monitor protein non-specific binding on the surface of microplate wells and evaluate the performance of blocking agents, there is still a great need to develop new methods to achieve the same goal from a new perspective. In this study, an innovative whole area scanning (WAS)-enabled direct-counting strategy was developed and validated through studying the blocking efficiency of different blocking agents on the non-specific binding of streptavidin-alkaline phosphatase conjugate (Strep-ALP, a model protein) to the surface of 96-well microplates. After non-specific binding of Strep-ALP in wells with or without blocking agents' treatment and loading of ELF™ 97 phosphate (ELFP), ALP in Strep-ALP conjugates converts ELFP to water-insoluble ELF™ 97 alcohol (ELFA), which precipitates locally, self-assembles into large needle structures, and glows green fluorescence upon excitation. After quenching the reaction, WAS of the whole wells allows us to directly count the number of individual fluorescent precipitates, which can be used to calculate and compare the blocking efficiency of three commonly used blocking agents (BSA, casein, and dry milk) based on mitigating the non-specific binding of Strep-ALP. WAS-enabled counting of individual needle-type precipitates opens a new avenue to investigate protein-solid surface binding as well as the efficiency of blocking agents with high sensitivity.

Visual detection of alkaline phosphatase based on ascorbic acid-triggered gel-sol transition of alginate hydrogel

Stimuli-responsive hydrogel has been emerged as a popular tool for chemical sensing due to its unique mechanical properties. In this work, we fabricated an ascorbic acid (AA)-responsive alginate hydrogel for the visual detection of alkaline phosphatase (ALP). This alginate hydrogel (RhB@Alg/Fe3+) was crosslinked with Fe3+, and rhodamine B (RhB) was encapsulated into the hydrogel as an indicating reagent to assistant visual detection. Because of the weak affinity of Fe2+ to alginate, the presence of reductive AA can trigger the dissolution of RhB@Alg/Fe3+ to give an observable red color in the sol solution. On this basis, by using ascorbic acid 2-phosphate as a substrate of ALP, which can be hydrolyzed by ALP to produce AA, the gel-sol transition process of RhB@Alg/Fe3+ was further modulated by ALP. This finding leads to a simple visual method for ALP detection with a low detection limit of 0.37 mU/mL and an excellent selectivity over other proteins. Compared with conventional colorimetric assays, this visual sensor shows the distinct advantages of simple fabrication, cost-effectiveness and easy to implement. We believe that this study can provide a new insight into the fabrication of responsive alginate hydrogel for promising applications in chemical sensing and biomedical fields.

Label-free monitoring of crystalline chitin hydrolysis by chitinase based on Raman spectroscopy.

We demonstrate a method for label-free monitoring of hydrolytic activity of crystalline-chitin-degrading enzyme, chitinase, by means of Raman spectroscopy. We found that crystalline chitin exhibited a characteristic Raman peak at 2995 cm-1, which did not appear in the reaction product, N,N'-diacetylchitobiose. We used this Raman peak as a marker of crystalline chitin degradation to monitor the hydrolytic activity of chitinase. When the crystalline chitin suspension and chitinase were mixed together, the peak intensity of crystalline chitin at 2995 cm-1 was linearly decreased depending on incubation time. The decrease in peak intensity was inversely correlated with the increase in the amount of released N,N'-diacetylchitobiose, which was measured by conventional colorimetric assay with alkaline ferricyanide. Our result, presented here, provides a new method for simple, in situ, and label-free monitoring of enzymatic activity of chitinase.

Evaluation of Inhibitory Effects of Caffeine on Human Carcinoma Cells

One of the world's most consumed medications is caffeine which is available in the vast majority of beverages. Previously, some effects of caffeine have been evaluated including its inhibitory effect on cancer cells. But, the influence of caffeine on esophagus carcinoma squamous cells (CSC) and head and neck carcinoma cells still has not well understood. Herein, the relation between different amounts of caffeine with the proliferation rate of human esophagus carcinoma squamous cell line KYSE-30 as well as human head and neck carcinoma cell line HN5 was evaluated. Furthermore, concentrations of caffeine were adjusted and their effect on cells were studied. The inhibitory effects of caffeine on cells were measured using the conventional colorimetric MTT assay after 3 and 7 day of incubation. Our findings are suggested that caffeine has a significant inhibitory effect on both cell lines at the concentrations of 20, 50, and 70 milli-mol (mmol). The result shows that caffeine can prevent the proliferation of carcinoma cells and it is a perfect candidate for therapeutic applications.

A Spheroid-Forming Hybrid Gold Nanostructure Platform That Electrochemically Detects Anticancer Effects of Curcumin in a Multicellular Brain Cancer Model.

In this study, a multifunctional platform that enables the highly efficient formation of 3D multicellular cancer spheroids and precise real-time assessments of the anticancer effects of curcumin in a brain tumor coculture model is reported. A highly conductive gold nanostructure (HCGN) is fabricated to facilitate cancer spheroid formation without using anti-cell adhesion molecules. A neuroblastoma (SH-SY5Y) and glioblastoma (U-87MG) coculture model is generated on HCGN with a specific cell-to-cell ratio (SH-SY5Y: U-87MG = 1:1), and their redox behaviors are successfully measured without destroying the distinct 3D structure of the multicellular spheroids. Using electrochemical signals as an indicator of spheroid viability, the effects of potential anticancer compounds on cocultured spheroids are further assessed. Remarkably, decreased cell viability in 3D spheroids caused by a low concentration of curcumin (30µM) is detectable using the electrochemical method (29.4%) but not with a conventional colorimetric assay (CCK-8). The detection is repeated more than ten times for both short- (63 h) and long-term cultivation (144 h) without damaging the spheroids, enabling real-time, non-destructive pharmacokinetic analysis of various drug candidates. Therefore, it can be concluded that the hybrid platform is a highly promising, precise, and high-throughput drug screening tool based on 3D cell cultivation.

Nitrosonaphthol reaction-assisted SERS assay for selective determination of 5-hydroxyindole-3-acetic acid in human urine

Urinary 5-hydroxyindole-3-acetic acid (5-HIAA) is a marker for diagnosis of patients with carcinoid tumors. In clinical practice, a simple colorimetric assay has widely been utilized for urinary 5-HIAA via its reaction with 1-nitroso-2-naphthol (NNa) in nitrous acid medium. However, this colorimetric assay has been criticized for lack of specificity. Herein, we proposed a novel SERS-based method for selective detection of urinary 5-HIAA by the use of a modified nitrosonaphthol reaction, in which hydrochloric acid is substituted for nitric acid to control the reaction conditions. This slight modification is crucial to be able to detect the cationic products in the acidic reaction mixture, because the chloride-aggregated Ag colloids not only create enormous hot spots for SERS enhancement, but also improve the chemical stability of nanostructured Ag substrates in acidic environment. Under optimized conditions, the present method can detect 5-HIAA within 12 min at concentrations as low as 1.2 ng/mL, which is 1000 times lower than that of conventional colorimetric method. And more importantly, the present method exhibits specific response toward 5-HIAA against other metabolites with similar structures in the urine. The relative standard deviation (RSD) of the present method is less than 5%, suggesting the acceptable reproducibility. The recoveries ranging from 89.6% to 106.3% were obtained for spiked human urine samples with RSD of 3.7–4.9%. Furthermore, several healthy person’s urine samples were also analyzed using the present method, and experimental results are in compliance with the levels recorded in a healthy population. On the basis of these results we can conclude that the present SERS-based method can provide a valuable alternative to conventional colorimetric assay for clinical diagnosis, evaluating prognosis, and monitoring of treatment in carcinoid tumors.

Quantitative Image-Based Cell Viability (QuantICV) Assay for Microfluidic 3D Tissue Culture Applications.

Microfluidic 3D tissue culture systems are attractive for in vitro drug testing applications due to the ability of these platforms to generate 3D tissue models and perform drug testing at a very small scale. However, the minute cell number and liquid volume impose significant technical challenges to perform quantitative cell viability measurements using conventional colorimetric or fluorometric assays, such as MTS or Alamar Blue. Similarly, live-dead staining approaches often utilize metabolic dyes that typically label the cytoplasm of live cells, which makes it difficult to segment and count individual cells in compact 3D tissue cultures. In this paper, we present a quantitative image-based cell viability (QuantICV) assay technique that circumvents current challenges of performing the quantitative cell viability assay in microfluidic 3D tissue cultures. A pair of cell-impermeant nuclear dyes (EthD-1 and DAPI) were used to sequentially label the nuclei of necrotic and total cell populations, respectively. Confocal microscopy and image processing algorithms were employed to visualize and quantify the cell nuclei in the 3D tissue volume. The QuantICV assay was validated and showed good concordance with the conventional bulk MTS assay in static 2D and 3D tumor cell cultures. Finally, the QuantICV assay was employed as an on-chip readout to determine the differential dose responses of parental and metastatic 3D oral squamous cell carcinoma (OSCC) to Gefitinib in a microfluidic 3D culture device. This proposed technique can be useful in microfluidic cell cultures as well as in a situation where conventional cell viability assays are not available.

Electro-kinetically driven route for highly sensitive blood pathology on a paper-based device.

Enhancing the sensitivity of colorimetric detection in paper-devices is a quintessential step in achieving frugal diagnosis. Here, we demonstrate an effective way of improving the detection sensitivity of paper-based devices, as mediated by electro-kinetic mechanisms. By directly employing blood plasma, we investigate the electro-kinetic clustering of glucose, a neutral molecule in paper devices. Under the influence of uniform electric field, dispersed glucose gets accumulated in the paper strips. Due to the combination of EOF and electrophoretic migration, we achieve twofold increase in the colour intensity for both normal and diabetic samples. This approach is robust and possesses better sensitivity than conventional colorimetric assays and can be easily extended to other body fluid based diagnosis. These results may turn out to be of profound importance in improving the quality of pathological diagnosis in low-cost paper-based point-of-care devices deployed in resource-limited settings.

Affinity Purification and Functional Characterization of Dynamin-Related Protein 1.

Purification of dynamin-related proteins is complicated by their oligomeric tendencies. In this chapter, we describe an established purification regime to isolate the mitochondrial fission protein Drp1 using bacterial expression. Key attributes of dynamins include their ability to hydrolyze GTP and self-assemble into larger polymers under specific conditions. Therefore, the GTPase activity of Drp1 should be examined to confirm isolation of functional protein, and we describe a conventional colorimetric assay to assess enzyme activity. To determine the ability of Drp1 to self-assemble, we induce Drp1 polymerization through addition of a non-hydrolyzable GTP analogue. A sedimentation assay provides a quantitative measure of polymerization that complements a qualitative assessment through visualization of Drp1 oligomers using negative-stain electron microscopy (EM). Importantly, we highlight the caveats of affinity tags and the influence that these peptide sequences can have on Drp1 function given their proximity to functional domains.

Domain structures of mucosa-associated lymphoid tissue lymphoma translocation 1 protein for nuclear localization in oral carcinoma cells and the proliferation inhibition

Mucosa-associated lymphoid tissue lymphoma translocation 1 protein (MALT1) consisting of death domain, Ig-like domains and caspase-like domain is expressed in nucleus of oral carcinoma cells, and loss of the expression closely associates with disease progression and stimulates proliferation of the cells. However, nothing is known about the molecular backgrounds. In this study, eight constructs with different domain constitution of human MALT1 and six constructs were transiently and stably transfected into oral carcinoma cell lines, respectively. The immunoblot analysis showed that constructs containing caspase-like domain was expressed in nucleus and the domain-deleted constructs in cytoplasm. Immunocytochemistry of stably transfected HSC2 oral carcinoma cells confirmed the caspase-like domain-dependent nuclear localization. Involvement of domains in proliferation of stably transfected HSC2 cells was quantified by the real-time and conventional colorimetric assays. In contrast to suppression of the proliferation by full-length wild-type MALT1, any domain-deleted constructs enhanced the proliferation. Death domain construct without caspase-like domain suppressed the proliferation when it was localized in nucleus by ligating with the nuclear localization signal. These results demonstrate that nuclear localization of MALT1 in oral carcinoma cells depends on the presence of caspase-like domain and that death domain nuclear entity is responsible for MALT1 inhibition of oral carcinoma cell proliferation. Nuclear localization of death domain led by caspase-like domain may suppress oral carcinoma progression.

Rapid characterization of the activities of lignin-modifying enzymes based on nanostructure-initiator mass spectrometry (NIMS)

BackgroundProducing valuable fuels and chemicals from lignin is a key factor for making lignocellulosic biomass economically feasible; however, significant roadblocks exist due to our lack of detailed understanding of how lignin is enzymatically depolymerized and of the range of possible lignin fragments that can be produced. Development of suitable enzymatic assays for characterization of putative lignin active enzymes is an important step towards improving our understanding of the catalytic activities of relevant enzymes. Previously, we have successfully built an assay platform based on glycan substrates containing a charged perfluorinated tag and nanostructure-initiator mass spectrometry to study carbohydrate active enzymes, especially various glycosyl hydrolyses. Here, we extend this approach to develop a reliable and rapid assay to study lignin-modifying enzymes.ResultsTwo β-aryl ether bond containing model lignin dimer substrates, designed to be suitable for studying the activities of lignin-modifying enzymes (LMEs) by nanostructure-initiator mass spectrometry (NIMS), were successful synthesized. Small-angle neutron scattering experiments showed that these substrates form micelles in solution. Two LMEs, laccase from the polypore mushroom Trametes versicolor, and manganese peroxidase (MnP) from white rot fungus Nematoloma frowardii, were tested for catalytic activity against the two model substrates. We show that the reaction of laccase and MnP with phenolic substrate yields products that arise from the cleavage of the carbon–carbon single bond between the α-carbon and the adjacent aryl carbon, consistent with the mechanism for producing phenoxy radical as reaction intermediates. Reactions of the nonphenolic substrate with laccase, on the other hand, adopt a different pathway by producing an α-oxidation product; as well as the cleavage of the β-aryl ether bond. No cleavage of the carbon–carbon bond between the α-carbon and the aryl carbon was observed. To facilitate understanding of reaction kinetics, the reaction time course for laccase activity on the phenolic substrate (I) was generated by the simultaneous measurement of all products at different time points of the reaction. Withdrawal of only a small sample aliquot (0.2 μL at each time point) ensured minimum perturbation of the reaction. The time course can help us to understand the enzyme kinetics.ConclusionsA new assay procedure has been developed for studying lignin-modifying enzymes by nanostructure-initiator mass spectrometry. Enzyme assays of a laccase and a MnP on phenolic and nonphenolic β-aryl ether substrates revealed different primary reaction pathways due to the availability of the phenoxy radical intermediates. Our assay provides a wealth of information on bond cleavage events not available using conventional colorimetric assays and can easily be carried out in microliter volumes and the quantitative analysis of product formation and kinetics is rapidly achieved by NIMS. This is the first time that NIMS technology was applied to study the activities of lignin-modifying enzymes. Unlike other previous works, our use of amphiphilic guaiacylglycerol β-O-4 substrate (I) enables the formation of micelles. This approach helps avoid the re-polymerization of the resulting monomeric product. As a result, our assay can clearly demonstrate the degradation pathways of phenolic guaiacylglycerol β-O-4 type of molecules with laccase and MnP.

Thionine mediated para-sulfonatocalix[4]arene capped AuNPs multilayers for sensitive electrochemical detection of acetylcholinesterase activity

Both macrocyclic supramolecules and gold nanoparticles (AuNPs) play important roles in bioanalysis. For the former, molecular recognitions can be carried out adequately; while for the latter, good biocompatibility, high chemical stability, surface functionalization, and facile synthesis make them considerable building blocks for novel hybrid nanomaterials. Some hybrid nanomaterials have been used 2D solution state colorimetric assay. It is a challenge to hybrid AuNPs and macrocyclic supramolecules for the interface-based assays. Here, we report a thionine mediated para-sulfonatocalix[4]arene capped AuNPs (pSC4-AuNPs) multilayers signal amplify strategy, which integrates nanoassembly with hybrid macrocyclic supramolecules nanomaterials for the sensitive detection of acetylcholinesterase (AChE) activity, by taking the assay of a neurological diseases marker as an example. In this strategy, the target catalyzing acetylcholine (ATCh) into thiocholine (TCh), while TCh was self-assembled on the gold chip via an AuS bond. Thionine mediated nanoassemblies of pSC4-AuNPs for signal amplification occurring on the electrode interface. As a result, this strategy integrates the advantages of nanotechnology, supramolecular recognition techniques, signal amplification and electrochemical detection and provides a tool for monitoring enzyme. The whole procedure of this novel strategy is similar to hybrid the conventional colorimetric and electrochemical assay, inheriting the usability. But in comparison with colorimetric and electrochemical assay, the performance is greatly improved. The detection limit can be lowered to 2.4 pU/mL, making it possible to detect the target marker sensitively. Therefore, this approach could be further extended to the assays of other targets and may contribute to the development of disease diagnosis and therapy in the future.