Classification and functional origins of stereocomplementary alcohol dehydrogenases for asymmetric synthesis of chiral secondary alcohols: A review.

Alcohol dehydrogenases (ADHs) mediated biocatalytic asymmetric reduction of ketones have been widely applied in the synthesis of optically active secondary alcohols with highly reactive hydroxyl groups ligated to the stereogenic carbon and divided into (R)- and (S)-configurations. Stereocomplementary ADHs could be applied in the synthesis of both enantiomers and are increasingly accepted as the "first of choice" in green chemistry due to the high atomic economy, low environmental factor, 100 % theoretical yield, and high environmentally friendliness. Due to the equal importance of complementary alcohols, development of stereocomplementary ADHs draws increasing attention. This review is committed to summarize recent advance in discovery of naturally evolved and tailor-made stereocomplementary ADHs, unveil the molecular mechanism of stereoselective catalysis in views of classification and functional basis, and provide guidance for further engineering the stereoselectivity of ADHs for the industrial biosynthesis of chiral secondary alcohol of industrial relevance.

Structural elucidation, binding sites exploration and biological activities of bound phenolics from Radix Puerariae Thomsonii.

Radix puerariae thomsonii (RPT) contains many phenolics and exhibits various health benefits. Although the free phenolics in RPT have been identified, the composition and content of bound phenolics, which account for approximately 20% of the total phenolic content, remain unknown. In this study, 12 compounds were isolated and identified from RPT-bound phenolic extracts, of which 2 were novel and 6 were reported first in RPT. ORAC and PSC antioxidant activities of 12 compounds, as well as their effects on alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), α-glucosidase, and α-amylase were evaluated. Genistein exhibited the highest ORAC activity, while daidzin demonstrated superior PSC activity. Five compounds, including two new compounds, exhibited the ability to activate both ADH and ALDH. All the compounds except 4-hydroxyphenylacetic acid methyl ester and 2,4,4'-trihydroxydeoxybenzoin demonstrated inhibitory effects on α-glucosidase and α-amylase. Alkaline hydrolysis and stepwise enzymatic hydrolysis revealed that bound phenolics in RPT mainly exist within starch.

Precision Engineering of the Co-immobilization of Enzymes for Cascade Biocatalysis.

The design and orderly layered co-immobilization of multiple enzymes on resin particles remain challenging. In this study, the SpyTag/SpyCatcher binding pair was fused to the N-terminus of an alcohol dehydrogenase (ADH) and an aldo-keto reductase (AKR), respectively. A non-canonical amino acid (ncAA), p-azido-L-phenylalanine (p-AzF), as the anchor for covalent bonding enzymes, was genetically inserted into preselected sites in the AKR and ADH. Employing the two bioorthogonal counterparts of SpyTag/SpyCatcher and azide-alkyne cycloaddition for the immobilization of AKR and ADH enabled sequential dual-enzyme coating on porous microspheres. The ordered dual-enzyme reactor was subsequently used to synthesize (S)-1-(2-chlorophenyl)ethanol asymmetrically from the corresponding prochiral ketone, enabling the in situ regeneration of NADPH. The reactor exhibited a high catalytic conversion of 74 % and good reproducibility, retaining 80 % of its initial activity after six cycles. The product had 99.9 % ee, which that was maintained in each cycle. Additionally, the double-layer immobilization method significantly increased the enzyme loading capacity, which was approximately 1.7 times greater than that of traditional single-layer immobilization. More importantly, it simultaneously enabled both the purification and immobilization of multiple enzymes on carriers, thus providing a convenient approach to facilitate cascade biocatalysis.

Substitution of both histidines in the active site of yeast alcohol dehydrogenase 1 exposes underlying pH dependencies.

Histidine residues 44 and 48 in yeast alcohol dehydrogenase (ADH) bind to the coenzymes NAD(H) and contribute to catalysis. The individual H44R and H48Q substitutions alter the kinetics and pH dependencies, and now the roles of other ionizable groups in the enzyme were studied in the doubly substituted H44R/H48Q ADH. The substitutions make the enzyme more resistant to inactivation by diethyl pyrocarbonate, modestly improve affinity for coenzymes, and substantially decrease catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The pH dependencies for several kinetic parameters are shifted from pK values for wild-type ADH of 7.3-8.1 to values for H44R/H48Q ADH of 8.0-9.6, and are assigned to the water or alcohol bound to the catalytic zinc. It appears that the rate of binding of NAD+ is electrostatically favored with zinc-hydroxide whereas binding of NADH is faster with neutral zinc-water. The pH dependencies of catalytic efficiencies (V/EtKm) for ethanol oxidation and acetaldehyde reduction are similarly controlled by deprotonation and protonation, respectively. The substitutions make an enzyme that resembles the homologous horse liver H51Q ADH, which has Arg-47 and Gln-51 and exhibits similar pK values. In the wild-type ADHs, it appears that His-48 (or His-51) in the proton relay systems linked to the catalytic zinc ligands modulate catalytic efficiencies.

Insight into the structural characteristics of self-assembled liposome with epigallocatechin gallate/alcohol dehydrogenase.

In this study, the encapsulation and structural characteristics of the self-assembled liposome formed by epigallocatechin gallate (EGCG) and alcohol dehydrogenase (ADH) were studied. According to the results, EGCG significantly increased the catalytic activity of ADH with a 33.33 % activation rate and the liposomes were able to entrap EGCG-ADH with an effectiveness of 88.94 %. The self-assembled monolayers had nanometer-sized particles, and the excellent self-assembled system was demonstrated by the low PDI value and high surface absolute potential. The scanning electron microscope showed that the self-assembled liposome was honeycomb, groove-shaped, and rough. The spectroscopic results showed that EGCG-ADH complex was formed through hydrogen bond, which changed the secondary structure of the liposome, and verified EGCG-ADH liposome system was successfully prepared. In vitro digestion experiments showed that the gastrointestinal tolerance and antioxidant activity of EGCG-ADH liposomes were significantly higher than those of free EGCG-ADH.

Using Capillary Electrophoresis to Investigate Protein Conformational and Compositional Heterogeneity.

Detailed insights into protein structure/function relationships require robust characterization methodologies. Free-solution capillary electrophoresis (CE) is a unique separation technique which is sensitive to the conformation and/or composition of proteins, and therefore provides information on the heterogeneity of these properties. Three unrelated, conformationally/compositionally-altered proteins were separated by CE. An electrophoretic mobility distribution was determined for each protein along with its conformational and/or compositional heterogeneity. The CE results were compared with molar mass distributions obtained from size-exclusion chromatography coupled to light scattering (SEC-MALS). Bovine serum albumin multimers and two monomeric species were separated, highlighting variations in conformational/compositional heterogeneity among the multimers. Analysis of yeast alcohol dehydrogenase resolved two monomeric conformers and various tetrameric species, illustrating the impact of zinc ion removal and disulfide bond reduction on the protein's heterogeneity. The apo (calcium-free) and holo forms of bovine α-lactalbumin were separated and differences in the species' heterogeneity were measured; by contrast, the SEC-MALS profiles were identical. Comparative analysis of these structurally unrelated proteins provided novel insights into the interplay between molar mass and conformational/compositional heterogeneity. Overall, this study expands the utility of CE by demonstrating its capacity to discern protein species and their heterogeneity, properties which are not readily accessible by other analytical techniques.

Directed Evolution and Immobilization of Lactobacillus brevis Alcohol Dehydrogenase for Chemo-Enzymatic Synthesis of Rivastigmine.

Rivastigmine is one of the several pharmaceuticals widely prescribed for the treatment of Alzheimer's disease. However, its practical synthesis still faces many issues, such as the involvement of toxic metals and harsh reaction conditions. Herein, we report a chemo-enzymatic synthesis of Rivastigmine. The key chiral intermediate was synthesized by an engineered alcohol dehydrogenase from Lactobacillus brevis (LbADH). A semi-rational approach was employed to improve its catalytic activity and thermal stability. Several LbADH variants were obtained with a remarkable increase in activity and melting temperature. Exploration of the substrate scope of these variants demonstrated improved activities toward various ketones, especially acetophenone analogs. To further recycle and reuse the biocatalyst, one LbADH variant and glucose dehydrogenase were co-immobilized on nanoparticles. By integrating enzymatic and chemical steps, Rivastigmine was successfully synthesized with an overall yield of 66 %. This study offers an efficient chemo-enzymatic route for Rivastigmine and provides several efficient LbADH variants with a broad range of potential applications.

Theoretical Analysis of Selectivity Differences in Ketoreductases toward Aldehyde and Ketone Carbonyl Groups.

Lactobacillus kefir alcohol dehydrogenase (LkADH) and ketoreductase from Chryseobacterium sp. CA49 (ChKRED12) exhibit different chemoselectivity and stereoselectivity toward a substrate with both keto and aldehyde carbonyl groups. LkADH selectively reduces the keto carbonyl group while retaining the aldehyde carbonyl group, producing optically pure R-alcohols. In contrast, ChKRED12 selectively reduces the aldehyde group and exhibits low reactivity toward ketone carbonyls. This study investigated the structural basis for these differences and the role of specific residues in the active site. Molecular dynamics (MD) simulations and quantum chemical calculations were used to investigate the interactions between the substrate and the enzymes and the essential cause of this phenomenon. The present study has revealed that LkADH and ChKRED12 exhibit significant differences in the structure of their respective active pockets, which is a crucial determinant of their distinct chemoselectivity toward the same substrate. Moreover, residues N89, N113, and E144 within LkADH as well as Q151 and D190 within ChKRED12 have been identified as key contributors to substrate stabilization within the active pocket through electrostatic interactions and van der Waals forces, followed by hydride transfer utilizing the coenzyme NADPH. Furthermore, the enantioselectivity mechanism of LkADH has been elucidated using quantum chemical methods. Overall, these findings not only provide fundamental insights into the underlying reasons for the observed differences in selectivity but also offer a detailed mechanistic understanding of the catalytic reaction.

Engineering diaryl alcohol dehydrogenase KpADH reveals importance of retaining hydration shell in organic solvent tolerance.

Alcohol dehydrogenases (ADHs) are synthetically important biocatalysts for the asymmetric synthesis of chiral alcohols. The catalytic performance of ADHs in the presence of organic solvents is often important since most prochiral ketones are highly hydrophobic. Here, the organic solvent tolerance of KpADH from Kluyveromyces polyspora was semi-rationally evolved. Using tolerant variants obtained, meticulous experiments and computational studies were conducted to explore properties including stability, activity and kinetics in the presence of various organic solvents. Compared with WT, variant V231D exhibited 1.9-fold improvement in ethanol tolerance, while S237G showed a 6-fold increase in catalytic efficiency, a higher T 50 15 $$ {\mathrm{T}}_{50}^{15} $$ , as well as 15% higher tolerance in 7.5% (v/v) ethanol. Based on 3 × 100 ns MD simulations, the increased tolerance of V231D and S237G against ethanol may be ascribed to their enhanced ability in retaining water molecules and repelling ethanol molecules. Moreover, 6.3-fold decreased KM value of V231D toward hydrophilic ketone substrate confirmed its capability of retaining hydration shell. Our results suggest that retaining hydration shell surrounding KpADH is critical for its tolerance to organic solvents, as well as catalytic performance. This study provides useful guidance for engineering organic solvent tolerance of KpADH and other ADHs.

Structural and biochemical characterization of Arabidopsis alcohol dehydrogenases reveals distinct functional properties but similar redox sensitivity.

Alcohol dehydrogenases (ADHs) are a group of zinc-binding enzymes belonging to the medium-length dehydrogenase/reductase (MDR) protein superfamily. In plants, these enzymes fulfill important functions involving the reduction of toxic aldehydes to the corresponding alcohols (as well as catalyzing the reverse reaction, i.e., alcohol oxidation; ADH1) and the reduction of nitrosoglutathione (GSNO; ADH2/GSNOR). We investigated and compared the structural and biochemical properties of ADH1 and GSNOR from Arabidopsis thaliana. We expressed and purified ADH1 and GSNOR and determined two new structures, NADH-ADH1 and apo-GSNOR, thus completing the structural landscape of Arabidopsis ADHs in both apo- and holo-forms. A structural comparison of these Arabidopsis ADHs revealed a high sequence conservation (59% identity) and a similar fold. In contrast, a striking dissimilarity was observed in the catalytic cavity supporting substrate specificity and accommodation. Consistently, ADH1 and GSNOR showed strict specificity for their substrates (ethanol and GSNO, respectively), although both enzymes had the ability to oxidize long-chain alcohols, with ADH1 performing better than GSNOR. Both enzymes contain a high number of cysteines (12 and 15 out of 379 residues for ADH1 and GSNOR, respectively) and showed a significant and similar responsivity to thiol-oxidizing agents, indicating that redox modifications may constitute a mechanism for controlling enzyme activity under both optimal growth and stress conditions.

Reducing the vicissitudes of heterologous prochiral substrate catalysis by alcohol dehydrogenases through machine learning algorithms.

Alcohol dehydrogenases (ADHs) are popular catalysts for synthesizing chiral synthons a vital step for active pharmaceutical intermediate (API) production. They are grouped into three superfamilies namely, medium-chain (MDRs), short-chain dehydrogenase/reductases (SDRs), and iron-containing alcohol dehydrogenases. The former two are used extensively for producing various chiral synthons. Many studies screen multiple enzymes or engineer a specific enzyme for catalyzing a substrate of interest. These processes are resource-intensive and intricate. The current study attempts to decipher the ability to match different ADHs with their ideal substrates using machine learning algorithms. We explore the catalysis of 284 antibacterial ketone intermediates, against MDRs and SDRs to demonstrate a unique pattern of activity. To facilitate machine learning we curated a dataset comprising 33 features, encompassing 4 descriptors for each compound. Subsequently, an ensemble of machine learning techniques viz. Partial Least Squares (PLS) regression, k-Nearest Neighbors (kNN) regression, and Support Vector Machine (SVM) regression, was harnessed. Moreover, the assimilation of Principal Component Analysis (PCA) augmented precision and accuracy, thereby refining and demarcating diverse compound classes. As such, this classification is useful for discerning substrates amenable to diverse alcohol dehydrogenases, thereby mitigating the reliance on high-throughput screening or engineering in identifying the optimal enzyme for specific substrate.

Origin of the enantioselectivity of alcohol dehydrogenase.

Alcohol dehydrogenases (ADH) are a family of enzymes that catalyse the interconversion between ketones/aldehydes and alcohols in the presence of NADPH cofactor. It is challenging to desymmetrise the substituted cyclopentane-1,3-dione by engineering an ADH, while the reaction mechanism of the metal independent ADH remains elusive. Here we measured the conversion of a model substrate 2-benzyl-2-methylcyclopentane-1,3-dione by LbADH and found it predominately gave the (2R,3R) product. Binding mode analysis of the substrate in LbADH from molecular dynamics simulations disclosed the origin of the enantioselectivity of the enzyme; the opening and closing of the loop 191-205 above the substrate are responsible for shaping the binding pocket to orientate the substrate, so as to give different stereoisomer products. Using QM/MM calculations, we elucidated the reaction mechanism of LbADH. Furthermore, we demonstrated the reaction profile corresponding to the production of different stereoisomers, which is in accordance with our experimental observations. This research here will shed a light on the rational engineering of ADH to achieve stereodivergent stereoisomer products.

Enhancing the activity of a monomeric alcohol dehydrogenase for site-specific applications by site-directed mutagenesis.

Gene fusion or co-immobilization are key tools to optimize enzymatic reaction cascades by modulating catalytic features, stability and applicability. Achieving a defined spatial organization between biocatalysts by site-specific applications is complicated by the involvement of oligomeric enzymes. It can lead to activity losses due to disturbances of the quaternary structures and difficulties in stoichiometric control. Thus, a toolkit of active and robust monomeric enzymes is desirable for such applications. In this study, we engineered one of the rare examples of monomeric alcohol dehydrogenases for improved catalytic characteristics by site-directed mutagenesis. The enzyme from the hyperthermophilic archaeon Thermococcus kodakarensis naturally exhibits high thermostability and a broad substrate spectrum, but only low activity at moderate temperatures. The best enzyme variants showed an ~5-fold (2-heptanol) and 9-fold (3-heptanol) higher activity while preserving enantioselectivity and good thermodynamic stability. These variants also exhibited modified kinetic characteristics regarding regioselectivity, pH dependence and activation by NaCl.

A pharmacophore-based approach to demonstrating the scope of alcohol dehydrogenases.

Barriers to the ready adoption of biocatalysis into asymmetric synthesis for early stage medicinal chemistry are addressed, using ketone reduction by alcohol dehydrogenase as a model reaction. An efficient substrate screening approach is used to show the wide substrate scope of commercial alcohol dehydrogenase enzymes, with a high tolerance to chemical groups employed in drug discovery (heterocycle, trifluoromethyl and nitrile/nitro groups) observed. We use our screening data to build a preliminary predictive pharmacophore-based screening tool using Forge software, with a precision of 0.67/1, demonstrating the potential for developing substrate screening tools for commercially available enzymes without publicly available structures. We hope that this work will facilitate a culture shift towards adopting biocatalysis alongside traditional chemical catalytic methods in early stage drug discovery.

Crosslinked Aggregates of Fusion Enzymes in Microaqueous Organic Media.

Baeyer-Villiger monooxygenases (BVMOs) are attractive for selectively oxidizing various ketones using oxygen into valuable esters and lactones. However, the application of BVMOs is restrained by cofactor dependency and enzyme instability combined with water-related downsides such as low substrate loading, low oxygen capacity, and water-induced side reactions. Herein, we described a redox-neutral linear cascade with in-situ cofactor regeneration catalyzed by fused alcohol dehydrogenase and cyclohexanone monooxygenase in aqueous and microaqueous organic media. The cascade conditions have been optimized regarding substrate concentrations as well as the amounts of enzymes and cofactors with the Design of Experiments (DoE). The carrier-free immobilization technique, crosslinked enzyme aggregates (CLEAs), was applied to fusion enzymes. The resultant fusion CLEAs were proven to function in microaqueous organic systems, in which the enzyme ratios, water contents (0.5-5 vol. %), and stability have been systematically studied. The fusion CLEAs showed promising operational (up to 5 cycles) and storage stability.

Industrial light at the end of the iron-containing (group III) alcohol dehydrogenase tunnel.

There are three prominent alcohol dehydrogenases superfamilies: short-chain, medium-chain, and iron-containing alcohol dehydrogenases (FeADHs). Many members are valuable catalysts for producing industrially relevant products such as active pharmaceutical intermediates, chiral synthons, biopolymers, biofuels, and secondary metabolites. However, FeADHs are the least explored enzymes among the superfamilies for commercial tenacities. They portray a conserved structure having a "tunnel-like" cofactor and substrate binding site with particular functions, despite representing high sequence diversity. Interestingly, phylogenetic analysis demarcates enzymes catalyzing distinct native substrates where closely related clades convert similar molecules. Further, homologs from various mesophilic and thermophilic microbes have been explored for designing a solvent and temperature-resistant enzyme for industrial purposes. The review explores different iron-containing alcohol dehydrogenases potential engineering of the enzymes and substrates helpful in manufacturing commercial products.

Dependence of crystallographic atomic displacement parameters on temperature (25-150†K) for complexes of horse liver alcohol dehydrogenase.

Enzymes catalyze reactions by binding and orienting substrates with dynamic interactions. Horse liver alcohol dehydrogenase catalyzes hydrogen transfer with quantum-mechanical tunneling that involves fast motions in the active site. The structures and B factors of ternary complexes of the enzyme with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol or NAD+ and 2,2,2-trifluoroethanol were determined to 1.1-1.3 Šresolution below the `glassy transition' in order to extract information about the temperature-dependent harmonic motions, which are reflected in the crystallographic B factors. The refinement statistics and structures are essentially the same for each structure at all temperatures. The B factors were corrected for a small amount of radiation decay. The overall B factors for the complexes are similar (13-16 Å2) over the range 25-100 K, but increase somewhat at 150 K. Applying TLS refinement to remove the contribution of pseudo-rigid-body displacements of coenzyme binding and catalytic domains provided residual B factors of 7-10 Å2 for the overall complexes and of 5-10 Å2 for C4N of NAD+ and the methylene carbon of the alcohols. These residual B factors have a very small dependence on temperature and include local harmonic motions and apparently contributions from other sources. Structures at 100 K show complexes that are poised for hydrogen transfer, which involves atomic displacements of ∼0.3 Šand is compatible with the motions estimated from the residual B factors and molecular-dynamics simulations. At 298 K local conformational changes are also involved in catalysis, as enzymes with substitutions of amino acids in the substrate-binding site have similar positions of NAD+ and pentafluorobenzyl alcohol and similar residual B factors, but differ by tenfold in the rate constants for hydride transfer.

Biochemical and Functional Characterization of an Iron-Containing Alcohol Dehydrogenase from Thermococcus barophilus Ch5.

Two iron-containing alcohol dehydrogenases (ADHs) are encoded in the genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba ADH641 and Tba ADH547). In our previous publication, we reported biochemical characteristics and catalytic mechanism of Tba ADH547. Herein, we present evidence that Tba ADH641 possesses two activities for ethanol oxidization and acetaldehyde reduction at high temperature, capable of using NAD(H) and NADP(H) as coenzyme. Biochemical data show that Tba ADH641 possesses optimal reaction temperature, thermostability, divalent ion requirement, and substrate specificity distinct from Tba ADH547 and other iron-containing ADH homologues. However, Tba ADH641 and Tba ADH547 display same optimal reaction pH. Kinetic analyses demonstrate that Tba ADH641 displays higher catalytic efficiency for acetaldehyde reduction than that for ethanol oxidation, which is consistent with Tba ADH547. Mutational data demonstrate that residues D115, K118, E159, D190, and E215 in Tba ADH641, which has not been described to date, are necessary for enzyme activity, thus augmenting our understanding on catalytic mechanism of iron-containing ADH. Overall, our work demonstrates that Tba ADH641 is an iron-containing ADH with novel features, which is distinct from Tba ADH547, thus providing a potential biocatalyst for biotransformation reaction.

Electrospun aluminum silicate nanofibers as novel support material for immobilization of alcohol dehydrogenase.

Ceramic materials with high surface area, large and open porosity are considered excellent supports for enzyme immobilization owing to their stability and reusability. The present study reports the electrospinning of aluminum silicate nanofiber supports from sol-gel precursors, the impact of different fabrication parameters on the microstructure of the nanofibers and their performance in enzyme immobilization. A change in nanofiber diameter and pore size of the aluminum silicate nanofibers was observed upon varying specific processing parameters, such as the sol-composition (precursor and polymer concentration), the electrospinning parameters and the subsequent heat treatment (calcination temperature). The enzyme, alcohol dehydrogenase (ADH), was immobilized on the aluminum silicate nanofibers by physical adsorption and covalent bonding. Activity retention of 17% and 42% was obtained after 12 d of storage and repeated reaction cycles for physically adsorbed and covalently bonded ADH, respectively. Overall, the immobilization of ADH on aluminum silicate nanofibers resulted in high enzyme loading and activity retention. However, as compared to covalent immobilization, a marked decrease in the enzyme activity during storage for physically adsorbed enzymes was observed, which was ascribed to leakage of the enzymes from the nanofibers. Such fibers can improve enzyme stability and promote a higher residual activity of the immobilized enzyme as compared to the free enzyme. The results shown in this study thus suggest that aluminum silicate nanofibers, with their high surface area, are promising support materials for the immobilization of enzymes.

Enzyme-embedded electrospun fiber sensor of hydrophilic polymer for fluorometric ethanol gas imaging in vapor phase.

Non-invasive measurement of volatile organic compounds (VOCs) emitted from living organisms is a powerful technique for diagnosing health conditions or diseases in humans. Bio-based gas sensors are suitable for the sensitive and selective measurement of a target VOC from a complex mixture of VOCs. Conventional bio-based sensors are normally prepared as wet-type probes to maintain proteins such as enzymes in a stable state, resulting in limitations in the commercialization of sensors, their operating environment, and performance. In this study, we present an enzyme-based fluorometric electrospun fiber sensor (eFES) mesh as a gas-phase biosensor in dry form. The eFES mesh targeting ethanol was fabricated by simple one-step electrospinning of polyvinyl alcohol with an alcohol dehydrogenase and an oxidized form of nicotinamide adenine dinucleotide. The enzyme embedded in the eFES mesh worked actively in a dry state without pretreatment. Substrate specificity was also maintained, and the sensor responded well to ethanol with a sufficient dynamic range. Adjustment of the pH and coenzyme quantity in the eFES mesh also affected enzyme activity. The dry-form biosensor-eFES mesh-will open a new direction for gas-phase biosensors because of its remarkable performance and simple fabrication, which is advantageous for commercialization.