Biosynthesis and applications of iron oxide nanocomposites synthesized by recombinant Escherichia coli.

Recombinant Escherichia coli (E. coli) strain that produces phytochelatin (PC) and/or metallothionein (MT) can synthesize various metal nanoparticles (NPs) by reducing metal ions. Here we report in vivo biosynthesis of iron oxide nanocomposites (NCs) using recombinant E. coli. We designed a strategy of biosynthesizing iron oxide NCs by first internalizing chemically synthesized iron oxide NPs, followed by the reduction of added metal ions on the surface of internalized NPs by PC and/or MT in E. coli. For this, chemically synthesized Fe3O4 NPs were internalized by recombinant E. coli, and then, Au and Ag ions were added for the biosynthesis of AuFe3O4 and AgFe3O4 NCs, respectively. The NCs synthesized were analyzed by transmission electron microscopy, UV-vis spectrophotometry, and X-ray diffractometry to characterize their shape, optical property, and crystallinity. The Fe3O4 NPs in the biosynthesized NCs allowed easy purification of the biosynthesized NCs by applying a magnetic field. The AuFe3O4 NCs were used for enzyme-linked immunosorbent assay to detect prostate-specific antigen protein, while AgFe3O4 NCs were utilized for the antimicrobial application with low minimum inhibitory concentration. As recombinant E. coli can uptake and reduce various NPs and metal ions, biosynthesis of a wide range of NCs as new nanomaterials will be possible for diverse applications. KEY POINTS: • AuFe3O4 and AgFe3O4 nanocomposites were synthesized by recombinant E. coli. • Escherichia coli synthesized different iron oxide NCs depending on the metal ions to be added. • Biosynthesized AuFe3O4 NC was used for ELISA and AgFe3O4 NC for antimicrobial tests.

Collagenase injection into the suprachoroidal space of the eye to expand drug delivery coverage and increase posterior drug targeting.

Injection into the suprachoroidal space (SCS) allows drug delivery targeted to sclera, choroid, and retina. Here, we studied SCS injection formulated with collagenase to expand drug delivery coverage and increase posterior drug targeting within SCS by breaking down collagen fibrils that link sclera and choroid in the SCS. When 1 µm latex microparticles were injected with a collagenase formulation using microneedles into the SCS of rabbit eyes ex vivo and incubated at 37 °C for 4 h, microparticle delivery coverage increased from 20% to 45% and enhanced posterior drug targeting. Collagenase concentration was optimized to 0.5 mg/mL to maximize expanded posterior delivery and minimize tissue damage. Effects of collagenase injection within SCS increased and then plateaued 4 h after injection. Simultaneous injection of collagenase and microparticles had a greater effect on expanded delivery in the SCS compared to sequential injection. Collagenase injection into the SCS of rabbit eyes in vivo was also effective to expand delivery and was generally well-tolerated, showing transiently lowered IOP, but no apparent lasting adverse effects on ocular tissues such as sclera, choroid, and retina, as determined by analyzing histology, sclera tensile strength, and fundus imaging. We conclude that addition of collagenase during SCS injection can expand drug delivery coverage and increase posterior drug targeting.

In Vivo Synthesis of Nanocomposites Using the Recombinant Escherichia coli.

Biogenic gold nanorod (AuNR)-Ag core-shell nanocomposites (NCs) are synthesized by using recombinant Escherichia coli to demonstrate in vivo synthesis of biogenic NCs for the first time. The chemically synthesized AuNRs are internalized into the E. coli, and Ag ions are reduced and grown on the surface of the AuNRs with the assistance of metal-binding proteins, producing biogenic core-shell AuNR-Ag NCs. The core-shell structure of the biogenic AuNR-Ag NC is confirmed by transmission electron microscopy and energy-dispersive X-ray analysis. The biogenic AuNR-Ag NCs exhibit good plasmonic effects. While the core-shell morphology of the AuNR and Ag NCs is due to the similar lattice of Au and Ag, the shape of the biogenic NCs composed of gold nanoparticles and Fe is aciniform, and that of Fe3 O4 NPs and Au/Ag is a network structure, demonstrating the controllability of biogenic nanosynthesis using diverse metal combinations with different NC morphologies.

Clinical and audiological characteristics of 1000Hz audiometric notch patients.

PURPOSE: Among the hearing loss patients, we can confirm that the hearing loss of the specific frequency decreases, such as the 2000Hz notch in otosclerosis and the 4000Hz notch (c5-dip) in noise-induced hearing loss. The 1000Hz notch (c3-dip), however, is rarely studied. We fortuitously encountered a group of patients with a 1kHz hearing loss and report it with a review of the literature. METHODS: Otological history, audiogram, diagnosis, occupation, and history of noise exposure were reviewed from charts and telephone interview, and compared between c3-dip and c5-dip patients (n=98). RESULTS: Thirty-one patients (mean age: 46.2years) demonstrated 1kHz hearing loss; these included 11 males. The pure-tone threshold was 37.97dB at 1kHz and the average threshold was 22.38dB at other frequencies. In the c3-dip group, tinnitus was the most common complaint, while sudden sensorineural hearing loss and idiopathic tinnitus (n=8 each) were the most common diagnoses. Female patients and unilateral cases were more common in the c3-dip than in the c5-dip group, and ear fullness was more common in the c3-dip group than in the c5-dip group. The duration of occupation-related noise exposure was longer in the c5 group, and head or ear trauma was more frequent in the c3-dip group. CONCLUSION: We have defined a new clinical entity of 1kHz hearing loss in patients, defined as the c3-dip, which was clinically and audiologically distinct from the c5-dip. Further study is needed to clarify this new entity of hearing loss.

Dynamic Covalent Assembly of Peptoid-Based Ladder Oligomers by Vernier Templating.

Dynamic covalent chemistry, in conjunction with template-directed assembly, enables the fabrication of extended nanostructures that are both precise and tough. Here we demonstrate the dynamic covalent assembly of peptoid-based molecular ladders with up to 12 rungs via scandium(III)-catalyzed imine metathesis by employing the principle of Vernier templating, where small precursor units with mismatched numbers of complementary functional groups are coreacted to yield larger structures with sizes determined by the respective precursor functionalities. Owing to their monomer diversity and synthetic accessibility, sequence-specific oligopeptoids bearing dynamic covalent pendant groups were employed as precursors for molecular ladder fabrication. The generated structures were characterized using matrix-assisted laser desorption/ionization mass spectrometry and gel permeation chromatography, confirming successful molecular ladder fabrication.

Combination of a Sample Pretreatment Microfluidic Device with a Photoluminescent Graphene Oxide Quantum Dot Sensor for Trace Lead Detection.

A novel trace lead ion (Pb(2+)) detection platform by combining a microfluidic sample pretreatment device with a DNA aptamer linked photoluminescent graphene oxide quantum dot (GOQD) sensor was proposed. The multilayered microdevice included a microchamber which was packed with cation exchange resins for preconcentrating metal ions. The sample loading and recovery were automatically actuated by a peristaltic polydimethylsiloxane micropump with a flow rate of 84 µL/min. Effects of the micropump actuation time, metal ion concentration, pH, and the volumes of the sample and eluent on the metal ion capture and preconcentration efficiency were investigated on a chip. The Pb(2+) samples whose concentrations ranged from 0.48 nM to 1.2 µM were successfully recovered with a preconcentration factor value between 4 and 5. Then, the preconcentrated metal ions were quantitatively analyzed with a DNA aptamer modified GOQD. The DNA aptamer on the GOQD specifically captured the target Pb(2+) which can induce electron transfer from GOQD to Pb(2+) upon UV irradiation, thereby resulting in the fluorescence quenching of the GOQD. The disturbing effect of foreign anions on the Pb(2+) detection and the spiked Pb(2+) real samples were also analyzed. The proposed GOQD metal ion sensor exhibited highly sensitive Pb(2+) detection with a detection limit of 0.64 nM and a dynamic range from 1 to 1000 nM. The on-chip preconcentration of the trace metal ions from a large-volume sample followed by the metal ion detection by the fluorescent GOQD sensor can provide an advanced platform for on-site water pollution screening.

A comparison of the effects of repetitive transcranial magnetic stimulation (rTMS) by number of stimulation sessions on hemispatial neglect in chronic stroke patients.

We investigated the effect of repetitive transcranial magnetic stimulation (rTMS) applied either during one session of stimulation, or by ten sessions of low-frequency stimulation over the left parietal cortex, on hemispatial neglect in stroke patients. We enrolled 34 subjects that had experienced a stroke. All subjects received 1,200 real rTMS over the left parietal cortex at an intensity of 90% of motor thresholds with 1 Hz. Subjects were divided into two groups. One group of subjects (n = 19) received real rTMS over the left parietal cortex in a single session of stimulation, and the other group (n = 15), underwent a total of ten sessions of daily stimulations for 2 weeks. Letter cancelation test, line bisection test, and Ota's task were administered to compare the effects of different rTMS protocols, before and after rTMS. The results showed no difference in baseline value between the single session group and the ten sessions group. Total ten sessions of low-frequency rTMS over the left parietal cortex, compared with the single session of rTMS, significantly improved hemispatial neglect in letter cancelation, line bisection, and Ota's task (P < 0.01). Finally, a total of ten sessions of low-frequency rTMS can be used in treatment by rTMS for patients suffering from hemispatial neglect after stroke.

Reconstructive surgery with an autologous bone graft in a dog with presumptive chronic non-bacterial osteomyelitis.

A 15-year-old Cocker Spaniel was referred to for the evaluation of left forelimb lameness. Radiographic and computed tomography examinations revealed osteolysis of the proximal left third, fourth and fifth metacarpal bones and pathological fractures of the proximal left fourth metacarpal bone. Histopathological examination via bone biopsy did not provide a definitive diagnosis, and the owner elected limb-sparing surgery. The fourth metacarpal bone and digits were amputated. Subsequently, autologous bone grafts were performed on the lytic area of the third and fifth metacarpal bones. The dog showed improvement in gait 7 weeks after reconstructive surgery. Chronic non-bacterial osteomyelitis (CNO) was diagnosed by exclusion. To the best of our knowledge, CNO has not been previously reported in dogs.

A Novel Passive Shoulder Exoskeleton Using Link Chains and Magnetic Spring Joints.

Work-related musculoskeletal disorders represent a major occupational disability issue, and 53.4% of these disorders occur in the back or shoulders. Various types of passive shoulder exoskeletons have been introduced to support the weight of the upper arm and work tools during overhead work, thereby preventing injuries and improving the work environment. The general passive shoulder exoskeleton is constructed with rigid links and joints to implement shoulder rotation, but there exists a challenge to align with the flexible joint movements of the human shoulder. Also, a force-generating part using mechanical springs require additional mechanical components to generate torque similar to the shoulder joint, resulting in increased overall volume and inertia to the upper arm. In this study, we propose a new type of passive shoulder exoskeleton that uses magnetic spring joint and link chain. The redundant degrees of freedom in the link chains enables to follow the shoulder joint movement in the horizontal direction, and the magnetic spring joint generates torque without additional parts in a compact form. Conventional exoskeletons experience a loss in the assisting torque when the center of shoulder rotation changed during arm elevation. Our exoskeleton minimizes the torque loss by customizing the installation height and initial angle of the magnetic spring joint. The performances of the proposed exoskeleton were verified by an electromyographic evaluation of shoulder-related muscles in overhead work and box lifting task.

Boosting the Output Performance of the MoS2 Monolayer-Based Piezoelectric Nanogenerator by Artificial Dual Strain Concentration.

Piezoelectric nanogenerators (PENGs) with molybdenum disulfide (MoS2) monolayers have been intensively studied owing to their superior mechanical durability and stability. However, the limited output performance resulting from a small active area and low strain levels continues to pose a significant challenge that should be overcome. Herein, we report a novel strategy for the epoch-making output performance of a PENG with a MoS2 monolayer by adopting the additive strain concentration concept. The simulation study indicates that strain in the MoS2 monolayer can be initially augmented by the wavy structure resulting from the prestretched poly(dimethylsiloxane) (PDMS) and is further increased through flexural deformation (i.e., bending). Based on these studies, we have developed concentrated strain-applied PENGs with MoS2 monolayers. The wavy structures effectively applied strain to the MoS2 monolayer and generated a piezoelectric output voltage and current of around 580 mV and 47.5 nA, respectively. Our innovative approach to enhancing the performance of PENGs with MoS2 monolayers through the artificial dual strain concept has led to groundbreaking results, achieving the highest recorded output voltage and current for PENGs based on two-dimensional (2D) materials, which provides unique opportunities for the 2D-based energy harvesting field and structural insight into how to improve the net strain on 2D materials.

Highly efficient microbial inactivation enabled by tunneling charges injected through two-dimensional electronics.

Airborne pathogens retain prolonged infectious activity once attached to the indoor environment, posing a pervasive threat to public health. Conventional air filters suffer from ineffective inactivation of the physics-separated microorganisms, and the chemical-based antimicrobial materials face challenges of poor stability/efficiency and inefficient viral inactivation. We, therefore, developed a rapid, reliable antimicrobial method against the attached indoor bacteria/viruses using a large-scale tunneling charge-motivated disinfection device fabricated by directly dispersing monolayer graphene on insulators. Free charges can be stably immobilized under the monolayer graphene through the tunneling effect. The stored charges can motivate continuous electron loss of attached microorganisms for accelerated disinfection, overcoming the diffusion limitation of chemical disinfectants. Complete (>99.99%) and broad-spectrum disinfection was achieved <1 min of attachment to the scaled-up device (25 square centimeters), reliably for 72 hours at high temperature (60°C) and humidity (90%). This method can be readily applied to high-touch surfaces in indoor environments for pathogen control.

Analyzing structure-photophysical property relationships for isolated T8, T10, and T12 stilbenevinylsilsesquioxanes.

Silsesquioxanes (SQs) are of considerable interest for hybrid electronic and photonic materials. However, to date, their photophysical properties have not been studied extensively, thus their potential remains conjecture. Here we describe the first known efforts to map structure-photophysical properties as a function of cage symmetry and size by comparing identically functionalized systems. Our focus here is on the solution photophysical properties of the title stilbenevinyl-SQs, which were characterized using single photon absorption, two-photon absorption, fluorescence emission, and fluorescence lifetime kinetics. We offer here the first detailed photophysical study of the larger pure T10 and T12 silsesquioxanes and show photophysical properties that differ as a function of size, especially in their fluorescence behavior, indicating that cage size and/or symmetry can strongly affect photophysical properties. We also find that they offer excitation-dependent emission (evidence of rare "red-edge" effects). The T10 stilbenevinyl-SQ offers up to a 10-fold increase in two-photon absorption cross section per chromophore over a free chromophore, signifying increased electronic coupling. The SQ cage compounds show "rise times" of 700-1000 fs and low anisotropy (~0.1) in fluorescence lifetime kinetic studies. These results indicate excited state energy transfer, unobserved for the free chromophores and unexpected for systems with "inert" silica cores and for 3-D hybrid molecular species. These findings provide the first detailed photophysical study of chromophore-functionalized T10 and T12 silsesquioxanes and show that SQs may be considered a separate class of compounds/materials with anticipated novel properties of value in developing new components for electronic and photonic applications.

A novel colorimetric immunoassay utilizing the peroxidase mimicking activity of magnetic nanoparticles.

A simple colorimetric immunoassay system, based on the peroxidase mimicking activity of Fe3O4 magnetic nanoparticles (MNPs), has been developed to detect clinically important antigenic molecules. MNPs with ca. 10 nm in diameter were synthesized and conjugated with specific antibodies against target molecules, such as rotaviruses and breast cancer cells. Conjugation of the MNPs with antibodies (MNP-Abs) enabled specific recognition of the corresponding target antigenic molecules through the generation of color signals arising from the colorimetric reaction between the selected peroxidase substrate, 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2. Based on the MNP-promoted colorimetric reaction, the target molecules were detected and quantified by measuring absorbance intensities corresponding to the oxidized form of TMB. Owing to the higher stabilities and economic feasibilities of MNPs as compared to horseradish peroxidase (HRP), the new colorimetric system employing MNP-Abs has the potential of serving as a potent immunoassay that should substitute for conventional HRP-based immunoassays. The strategy employed to develop the new methodology has the potential of being extended to the construction of simple diagnostic systems for a variety of biomolecules related to human cancers and infectious diseases, particularly in the realm of point-of-care applications.

Tolerability, acceptability, and reproducibility of topical STAR particles in human subjects.

Topical delivery to treat dermatological disease is constrained by low skin permeability to most drugs due to the stratum corneum barrier. STAR particles containing microneedle protrusions can be topically applied on the skin to create micropores that dramatically increase skin permeability, even to water-soluble compounds and macromolecules. This study addresses the tolerability, acceptability, and reproducibility of STAR particles rubbed on the skin at multiple pressures and after multiple applications to human subjects. One-time STAR particle application at pressures between 40 and 80 kPa showed that skin microporation and erythema directly correlated with increased pressure, and 83% of subjects reported STAR particles to be comfortable at all pressures. Repeated application of STAR particles for 10 consecutive days at 80 kPa showed that skin microporation (~0.5% of skin area), erythema (low-to-moderate), and comfort with self-administration (75%) were similar over the course of the study. Comfort of sensations associated with STAR particles increased from 58% to 71% during the study, and familiarity with STAR particles increased from 12.5% to 50% of subjects reporting STAR particle application not feeling different from other skin products. This study demonstrates that topically applied STAR particles were well tolerated and highly acceptable after application at various pressures and repeated daily use. These findings further suggest that STAR particles offer a safe and reliable platform to enhance cutaneous drug delivery.

Paper-Based Radial Flow Assay Integrated to Portable Isothermal Amplification Chip Platform for Colorimetric Detection of Target DNA.

A novel integrated detection system that introduces a paper-chip-based molecular detection strategy into a polydimethylsiloxane (PDMS) microchip and temperature control system was developed for on-site colorimetric detection of DNA. For the paper chip-based detection strategy, a padlock probe DNA (PLP)-mediated rolling circle amplification (RCA) reaction for signal amplification and a radial flow assay according to the Au-probe labeling strategy for visualization were optimized and applied for DNA detection. In the PDMS chip, the reactions for ligation of target-dependent PLP, RCA, and labeling were performed one-step under isothermal temperature in a single chamber, and one drop of the final reaction solution was loaded onto the paper chip to form a radial colorimetric signal. To create an optimal analysis environment, not only the optimization of molecular reactions for DNA detection but also the chamber shape of the PDMS chip and temperature control system were successfully verified. Our results indicate that a detection limit of 14.7 nM of DNA was achieved, and non-specific DNAs with a single-base mismatch at the target DNA were selectively discriminated. This integrated detection system can be applied not only for single nucleotide polymorphism identification, but also for pathogen gene detection. The adoption of inexpensive paper and PDMS chips allows the fabrication of cost-effective detection systems. Moreover, it is very suitable for operation in various resource-limited locations by adopting a highly portable and user-friendly detection method that minimizes the use of large and expensive equipment. Supplementary Information: The online version contains supplementary material available at 10.1007/s13206-023-00101-7.

3D porous sol-gel matrix incorporated microdevice for effective large volume cell sample pretreatment.

In this study, we demonstrated an effective sample pretreatment microdevice that could perform the capture, purification, and release of pathogenic bacteria with a large-volume sample and at a high speed and high-capture yield. We integrated a sol-gel matrix into the microdevice which forms three-dimensional (3D) micropores for the cell solution to pass through and provides a large surface area for the immobilization of antibodies to capture the target Staphylococcus aureus (S. aureus) cells. The antibody was linked to the surface of the sol-gel via a photocleavable linker, allowing the cell-captured antibody moiety to be released by UV irradiation. In addition to the optimization of the antibody immobilization and UV cleavage processes, the cell-capture efficiency was maximized by controlling the sample flow rate with a pumping scheme (3 steps, 5 steps: 3 steps with one flutter step, 7 steps: 3 steps with two flutter steps) and the pumping time (100, 200, and 300 ms). A quantitative capture analysis was performed by targeting a specific gene site of protein A of S. aureus in real-time PCR (RT-PCR). While the 3-step process with an actuation time of 100 ms showed the fastest flow rate (1 mL sample processing time in 10 min), the pumping scheme with the 7-step process and the 300 ms actuation time revealed the highest cell-capture efficiency. A limit of detection study with the 7-step and the 300 ms pumping scheme demonstrated that 100 cells per 100 µL were detected with a 70% yield, and even a single cell could be analyzed via on-chip sample preparation. Thus, our novel sol-gel based microdevice was proven more cost-effective, simple, and efficient in terms of its sample pretreatment ability compared to the use of a conventional 2D flat microdevice. This proposed sample pretreatment device can be further incorporated to an analytical functional unit to realize a micrototal analysis system (µTAS) with sample-in-answer-out capability in the fields of biomedical diagnostics, food safety testing, and environmental pollutant screening.

Sustainable Drug Release Using Nanoparticle Encapsulated Microneedles.

Microneedle (MN) is a minimally invasive drug delivery method that is directly inserted into the skin without pain to improve the efficiency of transdermal administration and is a drug delivery system used to treat various diseases. Furthermore, nanoparticle (NP)-based drug delivery methods suggest therapeutic strategies to improve drug solubility and increase drug delivery efficiency. Therefore, the drug delivery system in which NPs and MNs are integrated is a promising alternative to the existing delivery methods of poorly soluble and hydrophobic drugs and nucleic acid therapeutics. This system can increase the solubility of drugs and biocompatibility in the body and improve the therapeutic efficacy with sustained drug release. In this review, we investigated recent studies of NPs designed for drug delivery, sustained-release drug delivery MNs based on these NPs, and the applications for clinical treatments.

Six-month sustained delivery of anti-VEGF from in-situ forming hydrogel in the suprachoroidal space.

Patients with wet age-related macular degeneration (AMD) require intravitreal injections of bevacizumab (Bev) or other drugs, often on a monthly basis, which is a burden on the healthcare system. Here, we developed an in-situ forming hydrogel comprised of Bev and hyaluronic acid (HA) crosslinked with poly(ethylene glycol) diacrylate for slow release of Bev after injection into the suprachoroidal space (SCS) of the eye using a microneedle. Liquid Bev formulations were cleared from SCS within 5 days, even when formulated with high viscosity, unless Bev was conjugated to a high molecular-weight HA (2.6 MDa), which delayed clearance until 1 month. To extend release to 6 months, we synthesized in-situ forming Bev-HA hydrogel initially as a low-viscosity mixture suitable for injection and flow in the SCS to cover a large area extending to the posterior pole of the eye where the macula is located in humans. Within 1 h after injection, Bev and HA were crosslinked, which retained Bev for slow release as the hydrogel biodegraded. In vivo studies in the rabbit eye reported Bev release for >6 months, depending on gel formulation and Bev assay. The in-situ forming Bev-HA hydrogel was well tolerated, as assessed by clinical exam, fundus imaging, histological analysis, and intraocular pressure measurement. We conclude that Bev released from an in-situ forming hydrogel may enable long-acting treatments of AMD and other posterior ocular indications.

Evaluation of a Direct Reverse Transcription Loop-Mediated Isothermal Amplification Method without RNA Extraction (Direct RT-LAMP) for the Detection of Lymph Node Metastasis in Early Breast Cancer.

PURPOSE: Assessing lymph node metastasis, tumor-derived DNA, or tumor-derived RNA has previously been studied in place of immunohistochemical assay. Because a direct reverse transcription loop-mediated isothermal amplification method (direct RT-LAMP) has been previously developed in order to rapidly identify viruses in place of RNA extraction, our team hypothesized that a direct RT-LAMP assay can be employed as a substitute in order to detect tumor involvement of lymph nodes within breast cancer patients. MATERIALS AND METHODS: A total amount of 92 lymph nodes removed across 40 patients possessing breast cancer were collected at Kyungpook National University Chilgok Hospital between the months of November 2015 and February 2016. All samples were then evaluated and contrasted via both a direct RT-LAMP assay and routine histopathologic examination. RESULTS: The sensitivity and specificity of the direct RT-LAMP assay were 85.7% and 100%, respectively. The positive predictive value and negative predictive value were 100% and 94.4%, respectively. CONCLUSION: Direct RT-LAMP assay is capable of facilitating the detection of sentinel lymph node metastasis within breast cancer patients intraoperatively possessing an excellent sensitivity via a cost-effective and time-saving manner.

Precise Layer Control and Electronic State Modulation of a Transition Metal Dichalcogenide via Phase-Transition-Induced Growth.

Wafer-scale growth of transition metal dichalcogenides with precise control over the number of layers, and hence the electronic state is an essential technology for expanding the practical application of 2D materials. Herein, a new growth method, phase-transition-induced growth (PTG), is proposed for the precisely controlled growth of molybdenum disulfide (MoS2 ) films consisting of one to eleven layers with spatial uniformity on a 2 in. wafer. In this method, an energetically unstable amorphous MoSx Oy (a-MoSx Oy ) phase is effectively converted to a thermodynamically stable crystalline MoS2 film. The number of MoS2 layers is readily controlled layer-by-layer by controlling the amount of Mo atoms in a-MoSx Oy , which is also applicable for the growth of heteroatom-inserted MoS2 . The electronic states of intrinsic and Nb-inserted MoS2 with one and four layers grown by PTGare are analyzed based on their work functions. The work function of monolayer MoS2 effectively increases with the substitution of Nb for Mo. As the number of layers increases to four, charge screening becomes weaker, dopant ionization becomes easier, and ultimately the work function increases further. Thus, better electronic state modulation is achieved in a thicker layer, and in this respect, PTG has the advantage of enabling precise control over the film thickness.