Editorial

4 pages, 178 KiB

Open AccessEditorial

Enzyme-Based Biosensors and Their Applications

by Yu-Fan Fan, Zhao-Bin Guo and Guang-Bo Ge

Biosensors 2023, 13(4), 476; https://0-doi-org.brum.beds.ac.uk/10.3390/bios13040476 - 14 Apr 2023

Cited by 4 | Viewed by 2331

Enzymes constitute an extremely important class of biomacromolecules with diverse catalytic functions, which have been validated as key mediators for regulating cellular metabolism and maintaining homeostasis in living organisms [...] Full article

(This article belongs to the Special Issue Enzyme-Based Biosensors and Their Applications)

Research

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13 pages, 3073 KiB

Open AccessArticle

Functional Imaging and Inhibitor Screening of Human Pancreatic Lipase by a Resorufin-Based Fluorescent Probe

by Fan-Bin Hou, Na Zhang, Guang-Hao Zhu, Yu-Fan Fan, Meng-Ru Sun, Liang-Liang Nie, Guang-Bo Ge, Yue-Juan Zheng and Ping Wang

Biosensors 2023, 13(2), 283; https://0-doi-org.brum.beds.ac.uk/10.3390/bios13020283 - 16 Feb 2023

Cited by 3 | Viewed by 2015

Abstract

Human pancreatic lipase (hPL) is a crucial digestive enzyme responsible for the digestion of dietary lipids in humans, and inhibition of hPL is effective in reducing triglyceride intake, thereby preventing and treating obesity. In this study, a series of fatty acids with different [...] Read more.

Human pancreatic lipase (hPL) is a crucial digestive enzyme responsible for the digestion of dietary lipids in humans, and inhibition of hPL is effective in reducing triglyceride intake, thereby preventing and treating obesity. In this study, a series of fatty acids with different carbon chain lengths were constructed to the fluorophore resorufin based on the substrate preference of hPL. Among them, RLE was found to have the best combination of stability, specificity, sensitivity and reactivity towards hPL. Under physiological conditions, RLE can be rapidly hydrolyzed by hPL and released to resorufin, which triggered approximately 100-fold fluorescence enhancement at 590 nm. RLE was successfully applied for sensing and imaging of endogenous PL in living systems with low cytotoxicity and high imaging resolution. Moreover, a visual high-throughput screening platform was established using RLE, and the inhibitory effects of hundreds of drugs and natural products toward hPL were evaluated. Collectively, this study reports a novel and highly specific enzyme-activatable fluorogenic substrate for hPL that could serve as a powerful tool for monitoring hPL activity in complex biological systems and showcases the potential to explore physiological functions and rapid screening of inhibitors. Full article

(This article belongs to the Special Issue Enzyme-Based Biosensors and Their Applications)

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12 pages, 4925 KiB

Open AccessArticle

Construction and Manipulation of Serial Gradient Dilution Array on a Microfluidic Slipchip for Screening and Characterizing Inhibitors against Human Pancreatic Lipase

by Junqiang Yang, Yanyan Deng, Min Zhang, Shilun Feng, Sheng Peng, Shijia Yang, Peirong Liu, Gaozhe Cai and Guangbo Ge

Biosensors 2023, 13(2), 274; https://0-doi-org.brum.beds.ac.uk/10.3390/bios13020274 - 15 Feb 2023

Cited by 1 | Viewed by 2417

Abstract

Obesity is one of the foremost public health concerns. Human pancreatic lipase (hPL), a crucial digestive enzyme responsible for the digestion of dietary lipids in humans, has been validated as an important therapeutic target for preventing and treating obesity. The serial dilution technique [...] Read more.

Obesity is one of the foremost public health concerns. Human pancreatic lipase (hPL), a crucial digestive enzyme responsible for the digestion of dietary lipids in humans, has been validated as an important therapeutic target for preventing and treating obesity. The serial dilution technique is commonly used to generate solutions with different concentrations and can be easily modified for drug screening. Conventional serial gradient dilution is often performed with tedious multiple manual pipetting steps, where it is difficult to precisely control fluidic volumes at low microliter levels. Herein, we presented a microfluidic SlipChip that enabled formation and manipulation of serial dilution array in an instrument-free manner. With simple slipping steps, the compound solution could be diluted to seven gradients with the dilution ratio of 1:1 and co-incubated with the enzyme (hPL)-substrate system for screening the anti-hPL potentials. To ensure complete mixing of solution and diluent during continuous dilution, we established a numerical simulation model and conducted an ink mixing experiment to determine the mixing time. Furthermore, we also demonstrated the serial dilution ability of the proposed SlipChip using standard fluorescent dye. As a proof of concept, we tested this microfluidic SlipChip using one marketed anti-obesity drug (Orlistat) and two natural products (1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG) and sciadopitysin) with anti-hPL potentials. The IC₅₀ values of these agents were calculated as 11.69 nM, 8.22 nM and 0.80 μM, for Orlistat, PGG and sciadopitysin, respectively, which were consistent with the results obtained by conventional biochemical assay. Full article

(This article belongs to the Special Issue Enzyme-Based Biosensors and Their Applications)

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9 pages, 951 KiB

Open AccessCommunication

Electroanalytical Enzyme Biosensor Based on Cordia superba Enzyme Extract for the Detection of Phytomarkers in Kombucha

by Erica A. Batista, Marx O. A. Pereira, Isaac Y. L. Macêdo, Fabio B. Machado, Emily K. G. Moreno, Elgia P. Diniz, Italo G. V. Frazzão, Lorrayne S. C. Bernardes, Severino C. B. Oliveira and Eric S. Gil

Biosensors 2022, 12(12), 1112; https://0-doi-org.brum.beds.ac.uk/10.3390/bios12121112 - 01 Dec 2022

Cited by 1 | Viewed by 1265

Abstract

Antioxidants are responsible for many beneficial health effects and are highly present in natural products, such as kombucha. Biosensors’ development targeting antioxidants and phytomarkers are an active research field. This work aimed to propose a voltammetric polyphenolxidase (Cordia superba) biosensor for [...] Read more.

Antioxidants are responsible for many beneficial health effects and are highly present in natural products, such as kombucha. Biosensors’ development targeting antioxidants and phytomarkers are an active research field. This work aimed to propose a voltammetric polyphenolxidase (Cordia superba) biosensor for catechin and total phenolic compounds quantification in kombucha samples. Optimizations were performed on the biosensor of Cordia superba to improve the accuracy and selectivity, such as enzyme–substrate interaction time, analytical responses for different patterns and signal differences with the carbon paste and modified carbon paste electrode. Kombucha probiotic drink samples were fermented for 7 to 14 days at a controlled temperature (28 ± 2 °C). A linear curve was made for catechin with a range of 10.00 to 60.00 µM, with a limit of detection of 0.13 µM and limit of quantification of 0.39 µM. The biosensor proposed in this work was efficient in determining the patterns of phenolic compounds in kombucha. Full article

(This article belongs to the Special Issue Enzyme-Based Biosensors and Their Applications)

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0 pages, 2609 KiB

Open AccessArticle

A Catechol-Meter Based on Conventional Personal Glucose Meter for Portable Detection of Tyrosinase and Sodium Benzoate

by Tao Tian, Wei-Yi Zhang, Hang-Yu Zhou, Li-Jing Peng, Xi Zhou, Hao Zhang and Feng-Qing Yang

Biosensors 2022, 12(12), 1084; https://0-doi-org.brum.beds.ac.uk/10.3390/bios12121084 - 27 Nov 2022

Cited by 1 | Viewed by 2372

Abstract

In this study, the personal glucose meter (PGM) was first used as a fast and user-friendly meter for analyzing catechol (CA) based on the reduction of the mediator K₃[Fe(CN)₆] to K₄[Fe(CN)₆] in the glucose test [...] Read more.

In this study, the personal glucose meter (PGM) was first used as a fast and user-friendly meter for analyzing catechol (CA) based on the reduction of the mediator K₃[Fe(CN)₆] to K₄[Fe(CN)₆] in the glucose test strip. Then, an easy, low-cost, and convenient PGM-based method for detecting tyrosinase (TYR) activity and sodium benzoate (SBA) was developed on the basis of the TYR-catalyzed reaction. In this method, CA is oxidized to form o-benzoquinone by TYR, thereby reducing the residual amount of CA and the PGM readout. On the other hand, SBA can inhibit the oxidation of CA catalyzed by TYR and increase the residual amount of CA after the enzymatic reaction. Therefore, the activity of TYR is proportional to the difference in the PGM readout of CA, and the concentration of SBA is positively correlated with the residual amount of CA. After the relevant experimental conditions were systematically optimized, the proposed PGM-based method for the detection of TYR and SBA was successfully validated. The liner ranges are 1.0–103.3 U/mL and 6.25–1000 ppm, and the quantification limits are 1.0 U/mL and 6.25 ppm for TYR and SBA, respectively. Moreover, the spiked recovery tests in normal human serum and carbonate beverages (i.e., Cola, Sprite, and Fanta) were performed, and the recoveries (91.6–106.8%) further confirm the applicability of the PGM-based method in real sample analysis. Full article

(This article belongs to the Special Issue Enzyme-Based Biosensors and Their Applications)

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14 pages, 2215 KiB

Open AccessArticle

An Enzymatic Biosensor for the Detection of D-2-Hydroxyglutaric Acid in Serum and Urine

by Bo Wu, Zehua Li, Zepeng Kang, Chunling Ma, Haiyan Song, Fuping Lu and Zhiguang Zhu

Biosensors 2022, 12(2), 66; https://0-doi-org.brum.beds.ac.uk/10.3390/bios12020066 - 25 Jan 2022

Cited by 6 | Viewed by 3248

Abstract

D-2-hydroxyglutaric acid (D2HG) is overproduced as a result of the D-2-hydroxyglutaric aciduria and relevant cancers, caused by gene mutation. Accurate analysis of D2HG could help rapid diagnosis of these diseases and allow for timely treatment. In this work, a D-2-hydroxyglutarate dehydrogenase from Ralstonia [...] Read more.

D-2-hydroxyglutaric acid (D2HG) is overproduced as a result of the D-2-hydroxyglutaric aciduria and relevant cancers, caused by gene mutation. Accurate analysis of D2HG could help rapid diagnosis of these diseases and allow for timely treatment. In this work, a D-2-hydroxyglutarate dehydrogenase from Ralstonia solanacearum (RsD2HGDH) is cloned and recombinantly expressed. This enzyme features the direct electron transfer to chemical electron mediators (such as methylene blue (MB)) in the absence of additional coenzymes. Therefore, NAD⁺, a natural electron acceptor for the commercial D2HGDH and usually known for being unstable and difficult for immobilization can be avoided in the preparation of biosensors. The RsD2HGDH and MB are co-immobilized on a two-dimensional material, Ti₃C₂ MXene, followed by drop-coating on the gold screen-printed electrode (AuSPE) to construct a compact and portable biosensor. The D2HG in samples can be catalyzed by RsD2HGDH, where the current change is measured by chronoamperometry at −0.23 V. The biosensor shows a D2HG detection range of 0.5 to 120 µM (R² = 0.9974) with a sensitivity of 22.26 μA mM⁻¹ cm⁻² and a detection limit of 0.1 µM (S/N = 3). The biosensor retains 72.52% performance of its incipient state after 30 days of storage. The samples of D2HG-containing fetal bovine serum and artificial urine were analyzed with the recovery of 99.56% to 106.83% and 97.30% to 102.47% further indicating the great application potential of our portable D2HG biosensor. Full article

(This article belongs to the Special Issue Enzyme-Based Biosensors and Their Applications)

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13 pages, 2926 KiB

Open AccessArticle

Smartphone-Based Chemiluminescent Origami µPAD for the Rapid Assessment of Glucose Blood Levels

by Donato Calabria, Martina Zangheri, Ilaria Trozzi, Elisa Lazzarini, Andrea Pace, Mara Mirasoli and Massimo Guardigli

Biosensors 2021, 11(10), 381; https://0-doi-org.brum.beds.ac.uk/10.3390/bios11100381 - 09 Oct 2021

Cited by 19 | Viewed by 2723

Abstract

Microfluidic paper analytical devices (µPADs) represent one of the most appealing trends in the development of simple and inexpensive analytical systems for diagnostic applications at the point of care (POC). Herein, we describe a smartphone-based origami µPAD for the quantitative determination of glucose [...] Read more.

Microfluidic paper analytical devices (µPADs) represent one of the most appealing trends in the development of simple and inexpensive analytical systems for diagnostic applications at the point of care (POC). Herein, we describe a smartphone-based origami µPAD for the quantitative determination of glucose in blood samples based on the glucose oxidase-catalyzed oxidation of glucose leading to hydrogen peroxide, which is then detected by means of the luminol/hexacyanoferrate(III) chemiluminescent (CL) system. By exploiting the foldable µPAD format, a two-step analytical procedure has been implemented. First, the diluted blood sample was added, and hydrogen peroxide was accumulated, then the biosensor was folded, and a transport buffer was added to bring hydrogen peroxide in contact with CL reagents, thus promoting the CL reaction. To enable POC applicability, the reagents required for the assay were preloaded in the µPAD so that no chemicals handling was required, and a 3D-printed portable device was developed for measuring the CL emission using the smartphone’s CMOS camera. The µPAD was stable for 30-day storage at room temperature and the assay, displaying a limit of detection of 10 µmol L⁻¹, proved able to identify both hypoglycemic and hyperglycemic blood samples in less than 20 min. Full article

(This article belongs to the Special Issue Enzyme-Based Biosensors and Their Applications)

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Review

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22 pages, 9379 KiB

Open AccessReview

Spectrophotometric Assays for Sensing Tyrosinase Activity and Their Applications

by Yu-Fan Fan, Si-Xing Zhu, Fan-Bin Hou, Dong-Fang Zhao, Qiu-Sha Pan, Yan-Wei Xiang, Xing-Kai Qian, Guang-Bo Ge and Ping Wang

Biosensors 2021, 11(8), 290; https://0-doi-org.brum.beds.ac.uk/10.3390/bios11080290 - 23 Aug 2021

Cited by 22 | Viewed by 7211

Abstract

Tyrosinase (TYR, E.C. 1.14.18.1), a critical enzyme participating in melanogenesis, catalyzes the first two steps in melanin biosynthesis including the ortho-hydroxylation of L-tyrosine and the oxidation of L-DOPA. Previous pharmacological investigations have revealed that an abnormal level of TYR is tightly associated [...] Read more.

Tyrosinase (TYR, E.C. 1.14.18.1), a critical enzyme participating in melanogenesis, catalyzes the first two steps in melanin biosynthesis including the ortho-hydroxylation of L-tyrosine and the oxidation of L-DOPA. Previous pharmacological investigations have revealed that an abnormal level of TYR is tightly associated with various dermatoses, including albinism, age spots, and malignant melanoma. TYR inhibitors can partially block the formation of pigment, which are always used for improving skin tone and treating dermatoses. The practical and reliable assays for monitoring TYR activity levels are very useful for both disease diagnosis and drug discovery. This review comprehensively summarizes structural and enzymatic characteristics, catalytic mechanism and substrate preference of TYR, as well as the recent advances in biochemical assays for sensing TYR activity and their biomedical applications. The design strategies of various TYR substrates, alongside with several lists of all reported biochemical assays for sensing TYR including analytical conditions and kinetic parameters, are presented for the first time. Additionally, the biomedical applications and future perspectives of these optical assays are also highlighted. The information and knowledge presented in this review offer a group of practical and reliable assays and imaging tools for sensing TYR activities in complex biological systems, which strongly facilitates high-throughput screening TYR inhibitors and further investigations on the relevance of TYR to human diseases. Full article

(This article belongs to the Special Issue Enzyme-Based Biosensors and Their Applications)

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