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2.
Talanta ; 234: 122638, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34364447

RESUMO

Human hemoglobin (Hb) is a biomarker of several diseases, and monitoring of Hb levels is required during emergent surgery. However, rapid and sensitive Hb detection methods are yet to be developed. The present study established a rapid, convenient, and highly sensitive detection method for Hb in human serum using a bivalent antibody-enzyme complex (AEC). AECs are promising sensing elements because of their ability to bind specific targets and their catalytic activity that produce signals. We recently reported a convenient and universal method to fabricate bivalent AECs with two antibody fragments, using the SpyCatcher/SpyTag system. The present study applied a bivalent AEC for highly sensitive and quantitative detection of human Hb. The bivalent anti-Hb AEC was successfully prepared by incubating both N- and C-terminus SpyCatcher-fused glucose dehydrogenase and SpyTag-fused anti-Hb single-chain variable fragments at 4 °C. As expected, the bivalent AEC for Hb with a multimeric structure showed higher affinity than the monovalent AEC, by means of avidity effects, unlike that for soluble epidermal growth factor receptor with a monomeric structure; this contributed to a great improvement in sensitivity. Finally, we established a rapid and wash-free homogeneous electrochemical detection system for Hb by integrating magnetic beads. The linear range of the system completely covered the clinically required Hb levels, even in human serum. This technology provides an ideal point-of-care test for Hb and other multimeric biomarkers.


Assuntos
Anticorpos de Cadeia Única , Receptores ErbB , Hemoglobinas , Humanos
3.
Microb Cell Fact ; 20(1): 128, 2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34225717

RESUMO

BACKGROUND: The development of multiple gene expression systems, especially those based on the physical signals, such as multiple color light irradiations, is challenging. Complementary chromatic acclimation (CCA), a photoreversible process that facilitates the control of cellular expression using light of different wavelengths in cyanobacteria, is one example. In this study, an artificial CCA systems, inspired by type III CCA light-regulated gene expression, was designed by employing a single photosensor system, the CcaS/CcaR green light gene expression system derived from Synechocystis sp. PCC6803, combined with G-box (the regulator recognized by activated CcaR), the cognate cpcG2 promoter, and the constitutively transcribed promoter, the PtrcΔLacO promoter. RESULTS: One G-box was inserted upstream of the cpcG2 promoter and a reporter gene, the rfp gene (green light-induced gene expression), and the other G-box was inserted between the PtrcΔLacO promoter and a reporter gene, the bfp gene (red light-induced gene expression). The Escherichia coli transformants with plasmid-encoded genes were evaluated at the transcriptional and translational levels under red or green light illumination. Under green light illumination, the transcription and translation of the rfp gene were observed, whereas the expression of the bfp gene was repressed. Under red light illumination, the transcription and translation of the bfp gene were observed, whereas the expression of the rfp gene was repressed. During the red and green light exposure cycles at every 6 h, BFP expression increased under red light exposure while RFP expression was repressed, and RFP expression increased under green light exposure while BFP expression was repressed. CONCLUSION: An artificial CCA system was developed to realize a multiple gene expression system, which was regulated by two colors, red and green lights, using a single photosensor system, the CcaS/CcaR system derived from Synechocystis sp. PCC6803, in E. coli. The artificial CCA system functioned repeatedly during red and green light exposure cycles. These results demonstrate the potential application of this CCA gene expression system for the production of multiple metabolites in a variety of microorganisms, such as cyanobacteria.

4.
Nucleic Acids Res ; 49(11): 6069-6081, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34095949

RESUMO

Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin's peroxidase activity was discovered. Through our strategy-in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.


Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Luminol/metabolismo , Mioglobina/metabolismo , Peroxidase/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Simulação por Computador , Heme/metabolismo , Luminescência , Luminol/química , Ressonância Magnética Nuclear Biomolecular , Técnica de Seleção de Aptâmeros , Especificidade por Substrato
5.
Proc Natl Acad Sci U S A ; 118(25)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34161273

RESUMO

Enzymes are molecules that catalyze reactions critical to life. These catalysts are often studied in bulk water, where the influence of water volume on reactivity is neglected. Here, we demonstrate rate enhancement of up to two orders of magnitude for enzymes trapped in submicrometer water nanodroplets suspended in 1,2-dichloroethane. When single nanodroplets irreversibly adsorb onto an ultramicroelectrode surface, enzymatic activity is apparent in the amperometric current-time trace if the ultramicroelectrode generates the enzyme cofactor. Nanodroplet volume is easily accessible by integrating the current-time response and using Faraday's Law. The single nanodroplet technique allows us to plot the enzyme's activity as a function of nanodroplet size, revealing a strong inverse relationship. Finite element simulations confirm our experimental results and offer insights into parameters influencing single nanodroplet enzymology. These results provide a framework to profoundly influence the understanding of chemical reactivity at the nanoscale.

6.
Data Brief ; 36: 107028, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33948456

RESUMO

Aptamer is a nucleic acid ligand which specifically binds to its target molecule. Previously, we have designed an identification method of aptamer called "G-quadruplex (G4) promoter-derived aptamer selection (G4PAS)" [1]. In G4PAS procedure, putative G4 forming sequences (PQS) were explored in a promoter region of a target protein in human gene through computational analysis, and evaluated binding ability towards the gene product encoded in the downstream of the promoter. We investigated the topology of the obtained PQSs by circular dichroism measurement, as well as their binding ability against its target protein by surface plasmon resonance measurement and gel-shift assay. Additionally, the presence of nuclear localization signal in the target protein was predicted in silico. This data set summarized all the PQS sequences, their biochemical characteristics, and the presence of nuclear localization signal to address the possibility of binding of these PQS region to the target proteins in vivo. Those data should contribute to increase the success rate of G4PAS. Moreover, considering the G4 motifs in genomic DNA are suggested to be involved in vivo gene regulation [2], [3], this data set is also potentially beneficial for the cell biology field.

7.
Biosens Bioelectron ; 181: 113054, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33775474

RESUMO

Diabetes mellitus is a chronic illness in the United States affecting nearly 120 million adults, as well as increasing in children under the age of 18. Diabetes was also the 7th leading cause of death in the United States with 270 K deaths in 2017. Diabetes is best managed by tight glycemic control, as achieving near-normal glucose levels is key to reduce the risk of microvascular complications. Currently, continuous glucose monitoring (CGM) systems have been recognized as the ideal monitoring systems for glycemic control of diabetic patients. Briefly, a CGM system measures blood glucose levels in subcutaneous tissue by attaching a CGM sensor to the skin, allowing the users to make appropriate modifications to their medical interventions according to experience or empirically derived algorithms. The principles of the glucose sensing employed in the current commercially available CGM systems are mainly electrochemical, and employ the gold standard enzyme, glucose oxidase, as the glucose sensing molecule with the combination of hydrogen peroxide monitoring or with the combination of redox mediator harboring hydrogel. Recently, by employing an abiotic synthetic receptor harboring a fluorescent probe combined with a fluorescent detection system, a chronic CGM was commercialized. In addition, the development of less or non-invasive monitoring sensors targeting glucose in tears, sweat, saliva and urine have become of great interest although their clinical relevancy is still controversial. This review article introduces current and future technological aspects of CGM systems, the flagship technology in biosensor research, which was initiated, matured and is still growing in North America.


Assuntos
Técnicas Biossensoriais , Diabetes Mellitus Tipo 1 , Adulto , Glicemia , Automonitorização da Glicemia , Criança , Glucose , Humanos , Tecnologia
8.
Molecules ; 26(3)2021 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-33572552

RESUMO

Glycated albumin (GA) is an important glycemic control marker for diabetes mellitus. This study aimed to develop a highly sensitive disposable enzyme sensor strip for GA measurement by using an interdigitated electrode (IDE) as an electrode platform. The superior characteristics of IDE were demonstrated using one microelectrode of the IDE pair as the working electrode (WE) and the other as the counter electrode, and by measuring ferrocyanide/ferricyanide redox couple. The oxidation current was immediately reached at the steady state when the oxidation potential was applied to the WE. Then, an IDE enzyme sensor strip for GA measurement was prepared. The measurement of fructosyl lysine, the protease digestion product of GA, exhibited a high, steady current immediately after potential application, revealing the highly reproducible measurement. The sensitivity (2.8 nA µM-1) and the limit of detection (1.2 µM) obtained with IDE enzyme sensor strip were superior compared with our previously reported sensor using screen printed electrode. Two GA samples, 15 or 30% GA, corresponding to healthy and diabetic levels, respectively, were measured after protease digestion with high resolution. This study demonstrated that the application of an IDE will realize the development of highly sensitive disposable-type amperometric enzyme sensors with high reproducibility.


Assuntos
Técnicas Biossensoriais/instrumentação , Equipamentos Descartáveis , Enzimas/metabolismo , Albumina Sérica/análise , Eletrodos , Reprodutibilidade dos Testes
9.
Biosens Bioelectron ; 177: 112984, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33477030

RESUMO

Haemoglobin A1c (hemoglobin A1c, HbA1c) is an important long-term glycemic control marker for diabetes. The aim of this study was to develop an enzyme flow injection analysis (FIA) system using engineered fructosyl peptide oxidase (FPOx) based on 2.5th generation principle for an HbA1c automated analytical system. FPOx from Phaeosphaeria nodorum (PnFPOx) was engineered by introducing a Lys residue at the R414 position, to be modified with amine reactive phenazine ethosulfate (arPES) in proximity of FAD. The engineered PnFPOx mutant with minimized oxidase activity, N56A/R414K, showed quasi-direct electron transfer (quasi-DET) ability after PES-modification. The FIA system was constructed by employing a PES-modified PnFPOx N56A/R414K and operated at 0 V against Ag/AgCl. The system showed reproducible responses with a linear range of 20-500 µM for both fructosyl valine (FV) and fructosyl valylhistidine (FVH), with sensitivities of 0.49 nA µM-1 and 0.13 nA µM-1, and the detection limits of 1.3 µM and 2.0 µM for FV and FVH, respectively. These results indicate that the enzyme electrochemical FIA system covers the clinical range of HbA1c detection for more 200 consecutive measurements. Protease digested three different levels of HbA1c samples including healthy and diabetic range subjects were also measured with the FIA system. Thus, it will be possible to develop an integrated system consisting of sample pretreatment and sample electrochemical measurement based on an FIA system possessing quasi-DET type PnFPOx.


Assuntos
Técnicas Biossensoriais , Análise de Injeção de Fluxo , Ascomicetos , Elétrons , Hemoglobina A Glicada/análise , Humanos , Peptídeos
10.
Biosens Bioelectron ; 176: 112933, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33395570

RESUMO

The development of wearable multiplexed biosensors has been focused on systems to measure sweat l-lactate and other metabolites, where the employment of the direct electron transfer (DET) principle is expected. In this paper, a fusion enzyme between an engineered l-lactate oxidase derived from Aerococcus viridans, AvLOx A96L/N212K mutant, which is minimized its oxidase activity and b-type cytochrome protein was constructed to realize multiplexed DET-type lactate and glucose sensors. The sensor with a fusion enzyme showed DET to a gold electrode, with a limited operational range less than 0.5 mM. A mutation was introduced into the fusion enzyme to increase Km value and eliminate its substrate inhibition to construct "b2LOxS". Together with the employment of an outer membrane, the detection range of the sensor with b2LOxS was expanded up to 10 mM. A simultaneous lactate and glucose monitoring system was constructed using a flexible thin-film multiplexed electrodes with b2LOxS and a DET-type glucose dehydrogenase, and evaluated their performance in the artificial sweat. The sensors achieved simultaneous detection of lactate and glucose without cross-talking error, with the detected linear ranges of 0.5-20 mM for lactate and 0.1-5 mM for glucose, sensitivities of 4.1 nA/mM∙mm2 for lactate and 56 nA/mM∙mm2 for glucose, and limit of detections of 0.41 mM for lactate and 0.057 mM for glucose. The impact of the presence of electrochemical interferants (ascorbic acid, acetaminophen and uric acid), was revealed to be negligible. This is the first report of the DET-type enzyme based lactate and glucose dual sensing systems.


Assuntos
Técnicas Biossensoriais , Glicemia , Automonitorização da Glicemia , Eletrodos , Elétrons , Glucose , Glucose Oxidase , L-Lactato Desidrogenase
11.
Biosens Bioelectron ; 176: 112911, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33421758

RESUMO

A fusion enzyme composed of an Aspergillus flavus-derived flavin adenine dinucleotide glucose dehydrogenase (AfGDH) and an electron transfer domain of Phanerochaete chrysosporium-derived cellobiose dehydrogenase (Pcyb) was previously reported to show the direct electron transfer (DET) ability to an electrode. However, its slow intramolecular electron transfer (IET) rate from the FAD to the heme, limited the sensor signals. In this study, fusion FADGDH (Pcyb-AfGDH) enzymes were strategically redesigned by performing docking simulation, following surface-electrostatic potential estimation in the predicted area. Based on these predictions, we selected the amino acid substitution on Glu324, or on Asn408 to Lys to increase the positive charge at the rim of the interdomain region. Pcyb-AfGDH mutants were recombinantly produced using Pichia pastoris as the host microorganism, and their IET was evaluated. Spectroscopic observations showed that the Glu324Lys (E324K) and Asn408Lys (N408K) Pcyb-AfGDH mutants showed approximately 1.70- and 9.0-fold faster IET than that of wildtype Pcyb-AfGDH, respectively. Electrochemical evaluation revealed that the mutant Pcyb-AfGDH-immobilized electrodes showed higher DET current values than that of the wildtype Pcyb-AfGDH-immobilized electrodes at pH 6.5, which was approximately 9-fold higher in the E324K mutant and 15-fold higher in the N408K mutant, than in the wildtype. Glucose enzyme sensors employing N408K mutant was able to measure glucose concentration under physiological condition using artificial interstitial fluid at pH 7.4, whereas the one with wildtype Pcyb-AfGDH was not. These results indicated that the sensor employed the redesigned mutant Pcyb-AfGDH can be used for future continuous glucose monitoring system based on direct electron transfer principle. (247 words).


Assuntos
Técnicas Biossensoriais , Glucose 1-Desidrogenase , Glicemia , Automonitorização da Glicemia , Transporte de Elétrons , Elétrons , Flavina-Adenina Dinucleotídeo/metabolismo , Glucose , Glucose 1-Desidrogenase/metabolismo , Heme , Saccharomycetales
12.
Mar Biotechnol (NY) ; 23(1): 31-38, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32979137

RESUMO

In this study, we developed a green light-regulated T7 RNA polymerase expression system (T7 RNAP system), to provide a novel and versatile high-expression system for cyanobacteria without using any chemical inducer, realizing high expression levels comparable with previously reported for recombinant gene expression in cyanobacteria. The T7 RNAP system was constructed and introduced into Synechocystis sp. PCC6803. T7 RNAP was inserted downstream of the cpcG2 promoter, which is recognized and activated by the CcaS/CcaR two-component green-light-sensing system, to compose a vector plasmid, pKT-CS01, to achieve the induction of T7 RNAP expression only under green light illumination, with repression under red light illumination. The reporter gene, superfolder green fluorescent protein (sfGFP), was inserted downstream of the T7 promoter. Transcriptional analyses revealed that T7 RNAP was induced under green light but repressed under red light. Expression of the sfGFP protein derived from pKT-CS01 was observed under green light illumination and was approximately 10-fold higher than that in the control transformant, which expressed sfGFP directly under the cpcG2 promoter, which is directly regulated by CcaS/CcaR, under green light illumination. Comparison with the strong promoter expression systems Pcpc560 and PtrcΔlacO revealed that the expression of sfGFP by the T7 RNAP system was comparable with the levels obtained with strong promoters. These results demonstrated that the green light-regulated T7 RNAP gene expression system will be a versatile tool for future technological platform to regulate gene expression in cyanobacterial bioprocesses.

13.
Biosens Bioelectron ; 175: 112885, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33342691

RESUMO

Antibody-enzyme complexes (AECs) with binding ability to specific targets and catalytic activities to gain signals are known to be ideal sensing elements; however, AEC-based universal sensors applicable to point-of-care testing (POCT) have not yet been developed. Here, we achieved rapid and homogeneous electrochemical detection by fabricating a high-affinity bispecific AEC (bsAEC) using two Catcher/Tag systems. Recently, we reported a convenient and universal method to fabricate AECs using the SpyCatcher/SpyTag system. The resultant anti-epidermal growth factor receptor (anti-EGFR) AEC worked efficiently as a sensing element; however, the sensitivities did not meet the clinically required detection range of the soluble ectodomain of EGFR (sEGFR). To induce high affinity even to monomeric targets like sEGFR, we designed a convenient fabrication method for bsAEC using two Catcher/Tag systems, which did not express cross-reactivity. The anti-EGFR bsAEC was successfully prepared by constructing glucose dehydrogenase with two different catcher domains at the N- and C-terminus and by combining two corresponding Tag-fused anti-EGFR single-chain Fvs (scFvs), which recognize different epitopes on sEGFR. As expected, bsAEC showed a higher affinity than that of bivalent AEC with two identical anti-EGFR scFvs at low concentrations of sEGFR, and met the clinically required detection range of sEGFR. Further, by combining magnet beads, we established a rapid and wash-free homogeneous electrochemical detection method. This study offers new insights into the fabrication of universal POCT devices.


Assuntos
Anticorpos Biespecíficos , Técnicas Biossensoriais , Anticorpos de Cadeia Única , Epitopos , Receptores ErbB
14.
Biochem Biophys Res Commun ; 530(1): 82-86, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32828319

RESUMO

Fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) are the most popular and advanced enzymes for SMBG sensors because of their high substrate specificity toward glucose and oxygen insensitivity. However, this type of FADGDH hardly shows direct electron transfer (DET) ability. In this study, we developed a new DET-type FADGDH by harboring Cytochrome b562 (cyt b562) derived from Escherichia coli as the electron transfer domain. The structural genes encoding fusion enzymes composed of cyt b562 at either the N- or C-terminus of fungal FADGDH, (cyt b562-GDH or GDH-cyt b562), were constructed, recombinantly expressed, and characteristics of the fusion proteins were investigated. Both constructed fusion enzymes were successfully expressed in E. coli, as the soluble and GDH active proteins, showing cyt b562 specific redox properties. Thusconstructed fusion proteins showed internal electron transfer between FAD in FADGDH and fused cyt b562. Consequently, both cyt b562-GDH and GDH-cyt b562 showed DET abilities toward electrode. Interestingly, cyt b562-GDH showed much rapid internal electron transfer and higher DET ability than GDH-cyt b562. Thus, we demonstrated the construction and production of a new DET-type FADGDH using E.coli as the host cells, which is advantageous for future industrial application and further engineering.


Assuntos
Botrytis/genética , Grupo dos Citocromos b/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Glucose 1-Desidrogenase/genética , Botrytis/metabolismo , Grupo dos Citocromos b/metabolismo , Transporte de Elétrons , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Glucose 1-Desidrogenase/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato
15.
Sensors (Basel) ; 20(14)2020 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-32664558

RESUMO

Aptamer-based electrochemical sensors have gained attention in the context of developing a diagnostic biomarker detection method because of their rapid response, miniaturization ability, stability, and design flexibility. In such detection systems, enzymes are often used as labels to amplify the electrochemical signal. We have focused on glucose dehydrogenase (GDH) as a labeling enzyme for electrochemical detection owing to its high enzymatic activity, availability, and well-established electrochemical principle and platform. However, it is difficult and laborious to obtain one to one labeling of a GDH-aptamer complex with conventional chemical conjugation methods. In this study, we used GDH that was genetically fused to a DNA binding protein, i.e., zinc finger protein (ZF). Fused GDH can be attached to an aptamer spontaneously and site specifically in a buffer by exploiting the sequence-specific binding ability of ZF. Using such a fusion protein, we labeled a vascular endothelial growth factor (VEGF)-binding aptamer with GDH and detected the target electrochemically. As a result, upon the addition of glucose, the GDH labeled on the aptamer generated an amperometric signal, and the current response increased dependent on the VEGF concentration. Eventually, the developed electrochemical sensor proved to detect VEGF levels as low as 105 pM, thereby successfully demonstrating the concept of using ZF-fused GDH to enzymatically label aptamers.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Técnicas Eletroquímicas , Glucose 1-Desidrogenase , Fator A de Crescimento do Endotélio Vascular/análise , Humanos , Dedos de Zinco
16.
Int J Mol Sci ; 21(11)2020 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-32471202

RESUMO

In this review, recent progress in the engineering of the oxidative half-reaction of flavin-dependent oxidases and dehydrogenases is discussed, considering their current and future applications in bioelectrochemical studies, such as for the development of biosensors and biofuel cells. There have been two approaches in the studies of oxidative half-reaction: engineering of the oxidative half-reaction with oxygen, and engineering of the preference for artificial electron acceptors. The challenges for engineering oxidative half-reactions with oxygen are further categorized into the following approaches: (1) mutation to the putative residues that compose the cavity where oxygen may be located, (2) investigation of the vicinities where the reaction with oxygen may take place, and (3) investigation of possible oxygen access routes to the isoalloxazine ring. Among these approaches, introducing a mutation at the oxygen access route to the isoalloxazine ring represents the most versatile and effective strategy. Studies to engineer the preference of artificial electron acceptors are categorized into three different approaches: (1) engineering of the charge at the residues around the substrate entrance, (2) engineering of a cavity in the vicinity of flavin, and (3) decreasing the glycosylation degree of enzymes. Among these approaches, altering the charge in the vicinity where the electron acceptor may be accessed will be most relevant.


Assuntos
Transporte de Elétrons , Flavoproteínas/química , Oxirredutases/química , Animais , Flavinas/química , Flavinas/metabolismo , Flavoproteínas/metabolismo , Humanos , Oxirredutases/metabolismo , Oxigênio/química , Oxigênio/metabolismo
17.
Sensors (Basel) ; 20(10)2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32429321

RESUMO

In this paper, a novel electron mediator, 1-methoxy-5-ethyl phenazinium ethyl sulfate (mPES), was introduced as a versatile mediator for disposable enzyme sensor strips, employing representative flavin oxidoreductases, lactate oxidase (LOx), glucose dehydrogenase (GDH), and fructosyl peptide oxidase (FPOx). A disposable lactate enzyme sensor with oxygen insensitive Aerococcus viridans-derived engineered LOx (AvLOx), with A96L mutant as the enzyme, was constructed. The constructed lactate sensor exhibited a high sensitivity (0.73 ± 0.12 µA/mM) and wide linear range (0-50 mM lactate), showings that mPES functions as an effective mediator for AvLOx. Employing mPES as mediator allowed this amperometric lactate sensor to be operated at a relatively low potential of +0.2 V to 0 V vs. Ag/AgCl, thus avoiding interference from uric acid and acetaminophen. The lactate sensors were adequately stable for at least 48 days of storage at 25 °C. These results indicated that mPES can be replaced with 1-methoxy-5-methyl phenazinium methyl sulfate (mPMS), which we previously reported as the best mediator for AvLOx-based lactate sensors. Furthermore, this study revealed that mPES can be used as an effective electron mediator for the enzyme sensors employing representative flavin oxidoreductases, GDH-based glucose sensors, and FPOx-based hemoglobin A1c (HbA1c) sensors.


Assuntos
Aerococcus/enzimologia , Aminoácido Oxirredutases/química , Técnicas Biossensoriais , Elétrons , Glucose Desidrogenase/química , Oxigenases de Função Mista/química , Ésteres do Ácido Sulfúrico/química
18.
Int J Mol Sci ; 21(3)2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-32046321

RESUMO

Glucose oxidase (GOx) has been widely utilized for monitoring glycemic levels due to its availability, high activity, and specificity toward glucose. Among the three generations of electrochemical glucose sensor principles, direct electron transfer (DET)-based third-generation sensors are considered the ideal principle since the measurements can be carried out in the absence of a free redox mediator in the solution without the impact of oxygen and at a low enough potential for amperometric measurement to avoid the effect of electrochemically active interferences. However, natural GOx is not capable of DET. Therefore, a simple and rapid strategy to create DET-capable GOx is desired. In this study, we designed engineered GOx, which was made readily available for single-step modification with a redox mediator (phenazine ethosulfate, PES) on its surface via a lysine residue rationally introduced into the enzyme. Thus, PES-modified engineered GOx showed a quasi-DET response upon the addition of glucose. This strategy and the obtained results will contribute to the further development of quasi-DET GOx-based glucose monitoring dedicated to precise and accurate glycemic control for diabetic patient care.


Assuntos
Técnicas Biossensoriais/métodos , Glicemia/análise , Glucose Oxidase/metabolismo , Fenazinas/metabolismo , Engenharia de Proteínas , Aspergillus niger/enzimologia , Técnicas Eletroquímicas , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Glucose Oxidase/genética
19.
Biosens Bioelectron ; 151: 111974, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31999581

RESUMO

The l-lactate oxidase (LOx) based lactate sensors are widely used for clinical diagnostics, sports medicine, and food quality control. However, dissolved oxygen interference and electroactive interferent effects are inherent issues of current lactate sensors. In this paper, a quasi-direct electron transfer (quasi-DET) type lactate sensor was developed using rationally engineered Aerococcus viridans LOx (AvLOx) modified with amine-reactive phenazine ethosulfate (PES). Since the modification of wild type AvLOx by PES did not result quasi-DET, engineered AvLOx with additional Lys residue was designed. The additional Lys residue was introduced by substituting residue locating on the surface of AvLOx, and within 20 Šof the isoalloxazine ring of FMN. Among several constructed mutants, Ala96Leu/Asn212Lys double mutant showed the highest dye-mediated dehydrogenase activity with negligible oxidase activity, showing quasi-DET properties after PES modification, when the enzyme was immobilized on screen printed carbon electrode. The constructed electrode did not show oxygen interference in cyclic voltammetric analysis and distinct catalytic current with 20 mM l-lactate. The sensor performance of a chronoamperometric l-lactate sensor employing PES modified Ala96Leu/Asn212Lys AvLOx, marked with linear range between 0 and 1 mM, with sensitivity of 13 µA/mM∙cm2, and a limit of detection of 25 µM for l-lactate. By applying -200 mV vs. Ag/AgCl, l-lactate could be monitored with negligible interference from 170 µM ascorbic acid, 1.3 mM acetaminophen, 1.4 mM uric acid or 20 mM glucose. These results indicated that a quasi-DET type lactate sensor was developed that did not suffer from the interference of oxygen and representative electroactive ingredient compounds.


Assuntos
Aerococcus/isolamento & purificação , Técnicas Biossensoriais , Ácido Láctico/isolamento & purificação , Oxigenases de Função Mista/química , Aerococcus/química , Catálise , Enzimas Imobilizadas/química , Glucose/química , Humanos , Ácido Láctico/química , Oxirredução
20.
Bioelectrochemistry ; 132: 107414, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31838457

RESUMO

The history of the development of glucose sensors goes hand-in-hand with the history of the discovery and the engineering of glucose-sensing enzymes. Glucose oxidase (GOx) has been used for glucose sensing since the development of the first electrochemical glucose sensor. The principle utilizing oxygen as the electron acceptor is designated as the first-generation electrochemical enzyme sensors. With increasing demand for hand-held and cost-effective devices for the "self-monitoring of blood glucose (SMBG)", second-generation electrochemical sensor strips employing electron mediators have become the most popular platform. To overcome the inherent drawback of GOx, namely, the use of oxygen as the electron acceptor, various glucose dehydrogenases (GDHs) have been utilized in second-generation principle-based sensors. Among the various enzymes employed in glucose sensors, GDHs harboring FAD as the redox cofactor, FADGDHs, especially those derived from fungi, fFADGDHs, are currently the most popular enzymes in the sensor strips of second-generation SMBG sensors. In addition, the third-generation principle, employing direct electron transfer (DET), is considered the most elegant approach and is ideal for use in electrochemical enzyme sensors. However, glucose oxidoreductases capable of DET are limited. One of the most prominent GDHs capable of DET is a bacteria-derived FADGDH complex (bFADGDH). bFADGDH has three distinct subunits; the FAD harboring the catalytic subunit, the small subunit, and the electron-transfer subunit, which makes bFADGDH capable of DET. In this review, we focused on the two representative glucose sensing enzymes, fFADGDHs and bFADGDHs, by presenting their discovery, sources, and protein and enzyme properties, and the current engineering strategies to improve their potential in sensor applications.


Assuntos
Flavina-Adenina Dinucleotídeo/metabolismo , Glucose 1-Desidrogenase/metabolismo , Glucose/metabolismo , Engenharia de Proteínas , Elétrons , Fungos/enzimologia
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