RESUMO
To construct an electrochemical biosensing platform, we propose a glucose sensor whose electrode interface was modified by mesoporous silica (MPSi) as an electronic signal transmission interface between a biomarker and an electrochemical device. We develop an enzyme-free glucose sensor using an MPSi-coated Ta2O5 electrode in an actual biological fluid such as blood serum. MPSi includes a phenylboronic acid (PBA) molecule, in which glucose binds to a synthesized PBA-silane compound in an ca. 150 nm thick MPSi nanolayer, which changes the density of molecular charges of the PBA/glucose complex on the surface of MPSi. The charge changes derived from the equilibrium reaction of PBA with glucose lead to changes in surface potential of the Ta2O5 electrode, and the surface potential changes depending on glucose concentration were measured by a potentiometric detector. As a result, a remarkable surface potential response was observed in the vicinity of neutral pH. Kd = 6.0 mM and Vmax = 194 mV were obtained from the fitting curve of the Langmuir adsorption isotherm. Finally, we confirmed the glucose response of the PBA-MPSi-coated Ta2O5 substrate in human serum by considering the influence of various contaminants. Although the surface potential change was suppressed by approximately one-third of that in the buffer system, it was suggested that it could be applied to measurements in the blood glucose concentration range. From the results of this study, it was clarified that blood-level glucose response could be monitored using a PBA-MPSi-coated Ta2O5 substrate, which suggests the possibility of using a nonenzymatic glucose sensor as an alternative to the existing enzyme sensor.
Assuntos
Técnicas Biossensoriais , Dióxido de Silício , Eletrodos , Glucose , Humanos , Concentração de Íons de Hidrogênio , ÓxidosRESUMO
In this study, we report the label-free and reliable detection of allergic response using a cell-coupled gate field-effect transistor (cell-based FET). Rat basophilic leukemia (RBL-2H3) cells were cultured as a signal transduction interface to induce allergic reaction on the gate oxide surface of the FET, because IgE antibodies, which bind to Fcε receptors at the RBL-2H3 cell membrane, are specifically cross-linked by allergens, resulting in the allergic response of RBL-2H3 cells. In fact, the surface potential at the FET gate decreased owing to secretions such as histamine from the IgE-bound RBL-2H3 cells, which reacted with the allergen. This is because histamine, as one of the candidate secretions, shows basicity, resulting in a change in pH around the cell/gate interface. That is, the RBL-2H3-cell-based FET used in this study was originally from an ion-sensitive FET (ISFET), whose oxide surface (Ta2O5) with hydroxyl groups is fully responsive to pH on the basis of the equilibrium reaction. The allergic response of RBL-2H3 cells on the gate was also confirmed by estimating the amount of ß-hexosaminidase released together with histamine and was analyzed using the electrical properties based on an inflammatory response of secreted histamine with the vascular endothelial cell-based FET. Thus, the allergic responses were monitored in a nonoptical and real-time manner using the cell-based FETs with the cellular layers on the gate, which reproduced the in vivo system and were useful for the reliable detection of the allergic reaction.
Assuntos
Células Imobilizadas/metabolismo , Técnicas Eletroquímicas/métodos , Hipersensibilidade/diagnóstico , Transistores Eletrônicos , Animais , Linhagem Celular Tumoral , Células Imobilizadas/imunologia , Técnicas Eletroquímicas/instrumentação , Histamina/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina E/imunologia , Óxidos/química , Ratos , Tantálio/químicaRESUMO
In this study, we developed an ion-sensitive transparent-gate transistor (IS-TGT) for visible cell sensing. The gate sensing surface of the IS-TGT is transparent in a solution because a transparent amorphous oxide semiconductor composed of amorphous In-Ga-Zn-oxide (a-IGZO) with a thin SiO2 film gate that includes an indium tin oxide (ITO) film as the source and drain electrodes is utilized. The pH response of the IS-TGT was found to be about 56 mV/pH, indicating approximately Nernstian response. Moreover, the potential signals of the IS-TGT for sodium and potassium ions, which are usually included in biological environments, were evaluated. The optical and electrical properties of the IS-TGT enable cell functions to be monitored simultaneously with microscopic observation and electrical measurement. A platform based on the IS-TGT can be used as a simple and cost-effective plate-cell-sensing system based on thin-film fabrication technology in the research field of life science.
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Potássio/química , Sódio/química , Transistores Eletrônicos , Eletrodos , Gálio/química , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Índio/química , Íons/química , Óxidos/química , Tamanho da Partícula , Propriedades de Superfície , Compostos de Estanho/química , Células Tumorais Cultivadas , Zinco/químicaRESUMO
In this paper, we propose a highly sensitive and biocompatible glucose sensor using a semiconductor-based field effect transistor (FET) with a functionalized hydrogel. The principle of the FET device contributes to the easy detection of ionic charges with high sensitivity, and the hydrogel coated on the electrode enables the specific detection of glucose with biocompatibility. The copolymerized hydrogel on the Au gate electrode of the FET device is optimized by controlling the mixture ratio of biocompatible 2-hydroxyethylmethacrylate (HEMA) as the main monomer and vinylphenylboronic acid (VPBA) as a glucose-responsive monomer. The gate surface potential of the hydrogel FETs shifts in the negative direction with increasing glucose concentration from 10 µM to 40 mM, which results from the increase in the negative charges on the basis of the diol-binding of PBA derivatives with glucose molecules in the hydrogel. Moreover, the hydrogel coated on the gate suppresses the signal noise caused by the nonspecific adsorption of proteins such as albumin. The hydrogel FET can serve as a highly sensitive and biocompatible glucose sensor in in vivo or ex vivo applications such as eye contact lenses and sheets adhering to the skin.
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We developed a titanium-binding-peptide-1 (TBP-1)-tagged DNA polymerase, for self-oriented immobilization onto a titanium oxide (TiO2) substrate. The enzymatic function of a polymerase immobilized on a solid state device is strongly dependent on the orientation of the enzyme. The TBP-tagged DNA polymerase, which was derived from a hyperthermophilic archaeon, was designed to incorporate the RKLPDA peptide at the N-terminus, and synthesized by translation processes in Escherichia coli (E. coli). The specific binding of the TBP-tagged DNA polymerase onto a TiO2 substrate was clearly monitored by surface plasmon resonance spectroscopy (SPR) and by surface potential detection with an extended-gate field effect transistor (FET). In the SPR analyses, constant quantities of the DNA polymerase were stably immobilized on the titanium substrate under flow conditions, regardless of the concentration of the DNA polymerase, and could be completely removed by a 4 M MgCl2 wash after measurement. The FET signal showed the contribution of the molecular charge in the TBP motif to the binding with TiO2. In addition, the TBP-tagged DNA polymerase-tethered TiO2 gate electrode enabled the effective detection of the positive charges of hydrogen ions produced by the DNA extension reaction, according to the FET principle. Therefore, the self-oriented immobilization platform based on the motif-inserted enzyme is suitable for the quick and stable immobilization of functional enzymes on biosensing devices.
Assuntos
DNA Polimerase Dirigida por DNA/química , Peptídeos/química , Titânio/químicaRESUMO
OBJECTIVE: The body's glucose concentration is influenced by carbohydrate intake, insulin-induced carbohydrate reduction, and hepatic glycogen breakdown induced by stress hormones. This study investigated the potential of employing glucose fluctuations as a measure of stress by examining the relationship between heart rate variability (HRV) data and glucose levels during sleep in healthy subjects. METHODS: In this cross-sectional study, a chest-worn electrocardiogram (ECG) and continuous glucose monitoring device (CGM) were respectively used to monitor the heart rate intervals and glucose fluctuations of five subjects (two males, three females) during sleep. A time-series correlation analysis was performed on the HRV data extracted from heart rate intervals and the corresponding glucose fluctuation data. RESULTS: The time-series analysis of ECG and CGM data collected from subjects during sleep (n = 25 nights) revealed a moderate negative correlation between glucose levels and HRV, with a cross-correlation coefficient of r = -0.453. CONCLUSION: Similar to HRV, changes in stress levels can be detected by observing glucose fluctuations, particularly during sleep when the impact of food intake can be eliminated. Our findings highlight a significant correlation between glucose levels and HRV, indicating that glucose fluctuations can be used as an indicator of autonomic nervous system activity in an exploratory study.
Assuntos
Automonitorização da Glicemia , Glucose , Masculino , Feminino , Humanos , Frequência Cardíaca/fisiologia , Estudos Transversais , Glicemia , Sono/fisiologiaRESUMO
Silicon nanowire (SiNW) biosensors have attracted a lot of attention due to their superior sensitivity. Recently, the dependence of biomolecule detection sensitivity on the nanowire (NW) width, number, and doping density has been partially investigated. However, the primary reason for achieving ultrahigh sensitivity has not been elucidated thus far. In this study, we designed and fabricated SiNW biosensors with different widths (10.8-155 nm) by integrating a complementary metal-oxide-semiconductor process and electron beam lithography. We aimed to investigate the detection limit of SiNW biosensors and reveal the critical effect of the 10-nm-scaled SiNW width on the detection sensitivity. The sensing performance was evaluated by detecting antiovalbumin immunoglobulin G (IgG) with various concentrations (from 6 aM to 600 nM). The initial thickness of the depletion region of the SiNW and the changes in the depletion region due to biomolecule binding were calculated. The basis of this calculation are the resistance change ratios as functions of IgG concentrations using SiNWs with different widths. The calculation results reveal that the proportion of the depletion region over the entire SiNW channel is the essential reason for high-sensitivity detection. Therefore, this study is crucial for an indepth understanding on how to maximize the sensitivity of SiNW biosensors.
Assuntos
Técnicas Biossensoriais , Nanofios , Silício , Imunoglobulina G , Óxidos , ImpressãoRESUMO
Herein, we integrated angle-scanning surface plasmon resonance (SPR) and angle-fixed SPR as a hybrid angular-interrogation SPR to enhance the sensing performance. Galvanometer-mirror-based beam angle scanning achieves a 100-Hz acquisition rate of both the angular SPR reflectance spectrum and the angle-fixed SPR reflectance, whereas the use of near-infrared light enhances the refractive index (RI) sensitivity, range, and precision compared with visible light. Simultaneous measurement of the angular SPR reflectance spectrum and angle-fixed SPR reflectance boosts the RI change range, RI resolution, and RI accuracy to 10-1-10-6 RIU, 2.24 × 10-6 RIU, and 5.22 × 10-6 RIU, respectively. The proposed hybrid SPR is a powerful tool for wide-dynamic-range RI sensing with various applications.
RESUMO
Rapid, sensitive detection of biomolecules is important for biosensing of infectious pathogens as well as biomarkers and pollutants. For example, biosensing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still strongly required for the fight against coronavirus disease 2019 (COVID-19) pandemic. Here, we aim to achieve the rapid and sensitive detection of SARS-CoV-2 nucleocapsid protein antigen by enhancing the performance of optical biosensing based on optical frequency combs (OFC). The virus-concentration-dependent optical spectrum shift produced by antigen-antibody interactions is transformed into a photonic radio-frequency (RF) shift by a frequency conversion between the optical and RF regions in the OFC, facilitating rapid and sensitive detection with well-established electrical frequency measurements. Furthermore, active-dummy temperature-drift compensation with a dual-comb configuration enables the very small change in the virus-concentration-dependent signal to be extracted from the large, variable background signal caused by temperature disturbance. The achieved performance of dual-comb biosensing will greatly enhance the applicability of biosensors to viruses, biomarkers, environmental hormones, and so on.
Assuntos
Técnicas Biossensoriais , COVID-19 , Vírus , Humanos , COVID-19/diagnóstico , SARS-CoV-2 , Teste para COVID-19 , Antígenos ViraisRESUMO
The COVID-19 pandemic has created urgent demand for rapid detection of the SARS-CoV-2 coronavirus. Herein, we report highly sensitive detection of SARS-CoV-2 nucleocapsid protein (N protein) using nanoparticle-enhanced surface plasmon resonance (SPR) techniques. A crucial plasmonic role in significantly enhancing the limit of detection (LOD) is revealed for exceptionally large gold nanoparticles (AuNPs) with diameters of hundreds of nm. SPR enhanced by these large nanoparticles lowered the LOD of SARS-CoV-2 N protein to 85 fM, resulting in the highest SPR detection sensitivity ever obtained for SARS-CoV-2 N protein.
Assuntos
Proteínas do Nucleocapsídeo de Coronavírus , Ouro/química , Nanopartículas Metálicas/química , SARS-CoV-2/química , Ressonância de Plasmônio de Superfície , Proteínas do Nucleocapsídeo de Coronavírus/análise , Proteínas do Nucleocapsídeo de Coronavírus/química , Fosfoproteínas/análise , Fosfoproteínas/químicaRESUMO
For enzyme-/antibody-free and label-free biosensing, a molecularly imprinted polymer (MIP)-based membrane with phenylboronic acid (PBA) molecules, which induces the change in the density of molecular charges based on the small biomolecule-PBA diol binding, has been demonstrated to be suitable for the bioelectrical interface of biologically coupled gate field-effect transistor (bio-FET) sensors. MIP-coated gate FET sensors selectively detect various small biomolecules such as glucose, dopamine, sialic acid, and oligosaccharides without using labeled materials. In particular, the well-controlled MIP film by surface-initiated atom transfer radical polymerization (SI-ATRP) contributes to the quantitative analysis of small biomolecule sensing, resulting in potentiometric Langmuir isotherm adsorption analysis by which the parameters such as the binding affinity between small biomolecules and MIP cavities are evaluated. Also, the output electrical signal of even a random MIP-coated gate FET sensor is quantitatively analyzed using the bi-Langmuir adsorption isotherm equation, showing the adsorption mechanism of small biomolecules onto the template-specific MIP membrane. Thus, a platform based on the MIP bioelectrical interface for the bio-FET sensor is suitable for an enzyme-/antibody-free and label-free biosensing system in the fields of clinical diagnostics, drug discovery, the food industry, and environmental research.
RESUMO
As critical factors affecting the sensing performance of silicon nanowire (SiNW) biosensors, the structure, functional interface, and detection target were analyzed and designed to improve sensing performance. For an improved understanding of the dependence of sensor structure on sensitivity, a simple theoretical analysis was proposed to predict the sensitivity of biosensors with different SiNW types, widths, and doping concentrations. Based on the theoretical analysis, a biosensor integrating optimized critical factors was designed and fabricated. Optimizations focusing on the following aspects are considered: (1) employing n-type SiNW and controlling the impurity doping concentration of SiNW at approximately 2 × 1016-6 × 1016 atoms/cm3 to obtain a suitable charge density, (2) minimizing the SiNW width to 16.0 nm to increase the surface area-to-volume ratio, (3) using a native oxide layer on SiNW as a gate insulator to transport the captured charge molecules closer to the SiNW surface, (4) modifying the SiNW surface by 2-aminoethylphosphonic acid coupling to form a high-density self-assembled monolayer for enhancing the stability bound molecules, and (5) functionalizing the SiNW with ovalbumin molecules for specifically capturing the target immunoglobulin G (IgG) molecules. The sensing performance was evaluated by detecting IgG with concentrations ranging from 6 aM to 600 nM and control experiments. The SiNW biosensor revealed ultrahigh sensitivity and specific detection of target IgG with a measured limit of detection of 6 aM. The integration of the critical SiNW biosensor factors provides a significant possibility of a rapid and ultrasensitive diagnosis of diseases at their early stages.
Assuntos
Técnicas Biossensoriais/métodos , Imunoglobulina G/análise , Nanofios/química , Silício/química , Ácido Aminoetilfosfônico/química , Animais , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Imunoglobulina G/imunologia , Limite de Detecção , Ovalbumina/imunologia , Coelhos , Propriedades de Superfície , Transistores EletrônicosRESUMO
A platform based on a highly selective and sensitive detection device functionalized with a well-designed artificial biointerface is required for versatile biosensors. We develop a molecularly imprinted polymer (MIP)-coated gate field-effect transistor (FET) biosensor for low-concentration glucose detection in biological fluid samples such as tears in an enzyme-free manner. The MIP includes glucose templates (GluMIP), in which glucose binds to vinylphenylboronic acid in the copolymerized membrane, resulting in the change in the density of molecular charges of the phenylboronic acid (PBA)/glucose complex. The FET biosensor can detect small biomolecules as long as biomolecular recognition events cause intrinsic changes in the density of molecular charges. As a result, the changes in the output voltage detected using the GluMIP-based FET sensor are fitted to the Langmuir adsorption isotherm equation at various concentrations of sugars, showing the low detection limit of 3 µM and the high sensitivity of 115 mV/decade from 100 µM to 4 mM glucose. On the basis of the equation, the stability constant ( Ka) of PBA with glucose is calculated and found to markedly increase to Ka = 1192 M-1, which is higher by a factor of a few hundreds than Ka = 4.6 M-1 obtained by nonelectrical detection methods. Moreover, the GluMIP-coated gate FET sensor shows an approximately 200-fold higher selectivity for glucose than for fructose. This is because glucose binds to PBA more selectively than fructose in the templates, resulting in the generation of negative charges. The electrical properties of the MIP-coated electrode are also evaluated by measuring capacitance. Our work suggests a new strategy of designing a platform based on the MIP-coated gate FET biosensor, which is suitable for a highly selective, sensitive, enzyme-free biosensing system.
Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Glucose/análise , Impressão Molecular , Ácidos Borônicos/química , EletrodosRESUMO
A highly sensitive catecholamine (CA) sensor was created using a biointerface layer composed of a biopolymer and a potentiometric detection device. For the detection of CAs, 3-aminophenylboronic acid (3-NH2-PBA) was reacted with the carboxyl side chain of l-3,4-dihydroxyphenylalanine (l-dopa, LD) and the PBA-modified l-dopa was directly copolymerized with LD on an Au electrode, resulting in a 3.5 nm thick PBA-modified poly(PBA-LD/LD) layer-coated Au electrode. By connecting the PBA-LD-coated Au electrode to a field-effect transistor (FET), the molecular charge changes at the biointerface of the Au electrode, which was caused by di-ester binding of the PBA-CA complex, were transduced into gate surface potential changes. Effective CAs included LD, dopamine (DA), norepinephrine (NE), and epinephrine (EP). The surface potential of the PBA-LD-coated Au changed after the addition of 40 nM of each CA solution; notably, the PBA-LD-coated Au showed a higher sensitivity to LD because the surface potential change could already be observed after 1 nM of LD was added. The fundamental parameter analyses of the PBA-LD to CA affinity from the surface potential shift against each CA concentration indicated the highest affinity to LD (binding constant (Ks): 1.68 × 106 M-1, maximum surface potential shift (Vmax): 182 mV). Moreover, the limit of detection for each CA was 3.5 nM in LD, 12.0 nM in DA, 7.5 nM in NE, and 12.6 nM in EP. From these results, it is concluded that the poly(PBA-LD/LD)-coated gate FET could become a useful biosensor for neurotransmitters, hormones, and early detection of Parkinson's disease.
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We report a well-designed biointerface enabling the selective and quantitative detection of dopamine (DA) using a potentiometric biosensor. To enhance the detection selectivity of DA, a DA-templated molecularly imprinted polymer (DA-MIP) was synthesized on the extended Au gate electrode of a field-effect transistor (FET) biosensor. For a quantitative DA analysis, the thickness of the DA-MIP was controlled to ca. 60â¯nm by surface-initiated atom transfer radical polymerization (SI-ATRP). In this process, the DA-MIP was copolymerized with vinylphenylboronic acid (vinyl-PBA), inducing molecular charges at the biointerface of the FET gate electrode. These charges were generated by the diol-binding between PBA and dopamine (a catecholamine), and were directly detected as a change in surface potential. In fact, the surface potential at the gate of the DA-MIP-coated FET responded significantly to DA added at concentrations ranging from 40â¯nM to 20⯵M, whereas that of a non-imprinted polymer (NIP)-coated FET hardly changed over this range. Moreover, by measuring the kinetic parameters and electrochemical properties of well-designed devices with various added catecholamines, we confirmed that the DA-MIP-coated FET biosensor selectively and quantitatively detects DA.
Assuntos
Técnicas Biossensoriais/métodos , Dopamina/análise , Polímeros/química , Técnicas Biossensoriais/instrumentação , Catecolaminas/análise , Catecolaminas/química , Dopamina/química , Técnicas Eletroquímicas/métodos , Desenho de Equipamento , Ouro/química , Impressão Molecular , Polimerização , Propriedades de Superfície , TermodinâmicaRESUMO
Surface-coated poly(catecholamine) (pCA) films have attracted attention as biomaterial interfaces owing to their biocompatible and physicochemical characteristics. In this paper, we report that pCA-film-coated electrodes are useful for potentiometric biosensing devices. Four different types of pCA film, l-dopa, dopamine, norepinephrine, and epinephrine, with thicknesses in the range of 7-27 nm were electropolymerized by oxidation on Au electrodes by using cyclic voltammetry. By using the pCA-film electrodes, the pH responsivities were found to be 39.3-47.7 mV/pH within the pH range of 1.68 to 10.01 on the basis of the equilibrium reaction with hydrogen ions and the functional groups of the pCAs. The pCA films suppressed nonspecific signals generated by other ions (Na+, K+, Ca2+) and proteins such as albumin. Thus, the pCA-film electrodes can be used in pH-sensitive and pH-selective biosensors. HeLa cells were cultivated on the surface of the pCA-film electrodes to monitor cellular activities. The surface potential of the pCA-film electrodes changed markedly because of cellular activity; therefore, the change in the hydrogen ion concentration around the cell/pCA-film interface could be monitored in real time. This was caused by carbon dioxide or lactic acid that is generated by cellular respiration and dissolves in the culture medium, resulting in the change of hydrogen concentration. pCA-film electrodes are suitable for use in biocompatible and pH-responsive biosensors, enabling the more selective detection of biological phenomena.
Assuntos
Técnicas Biossensoriais/métodos , Catecolaminas/química , Materiais Revestidos Biocompatíveis/química , Polímeros/química , Potenciometria/métodos , Técnicas Biossensoriais/instrumentação , Técnicas de Cultura de Células , Eletrodos , Ouro/química , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Potenciometria/instrumentação , Propriedades de SuperfícieRESUMO
The brown-rot basidiomycete Fomitopsis palustris produces a major extracellular enzyme of 72 kDa when the fungus is incubated in cellulose culture with 0.2% cellobiose. This protein was purified by column chromatography, and the amino acid sequences of its proteolytic fragments were analyzed. The N-terminal amino acid sequence of one of the fragments showed high identity with fungal glycoside hydrolase family 15 glucoamylases. As its kinetic efficiency increased in proportion to the degree of polymerization of the substrate, the protein was identified as a glucoamylase. A cDNA encoding the glucoamylase (gla) was cloned by reverse transcriptase PCR.
Assuntos
Glucana 1,4-alfa-Glucosidase/genética , Glucana 1,4-alfa-Glucosidase/isolamento & purificação , Polyporales/enzimologia , Polyporales/genética , Sequência de Aminoácidos , Aminoácidos/análise , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Fúngico/genética , Genes Fúngicos , Glucana 1,4-alfa-Glucosidase/metabolismo , Cinética , Dados de Sequência MolecularRESUMO
Cellobiose dehydrogenase (CDH) was purified from the brown-rot fungus Coniophora puteana grown in culture containing crystalline cellulose as a carbon source. The purified enzyme gave a single band at 115 kDa on SDS-PAGE and showed a typical flavocytochrome absorption spectrum. The enzyme oxidized both cellobiose and cellooligosaccharides, but not their monomer, glucose, suggesting typical kinetic features of CDH. A cDNA encoding CDH was cloned by RT-PCR using primers designed from the consensus sequences of known CDHs from white-rot fungi. The cDNA consists of 2448 bp, including an open reading frame encoding the 18 amino acids of the putative signal peptide and the 756 amino acids of the mature protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) data for tryptic fragments of the purified C. puteana CDH were consistent with partial amino acid sequences of the mature protein deduced from the cloned cDNA. Moreover, the sequences contained common characteristics of CDH, i.e., two possible residues for a heme ligand (Met 64 and His 160), a flavin-binding motif, and two glucose-methanol-choline oxidoreductase motifs. This is the first cloning of CDH from a brown-rot fungus, and the results suggest structural and kinetic similarity of C. puteana CDH to white-rot fungal CDHs.