Identification of Gluconacetobacter xylinus LYP25 and application to bacterial cellulose production in biomass hydrolysate with acetic acid.

Bacterial cellulose (BC) is a remarkable biomacromolecule with potential applications in food, biomedical, and other industries. However, the low economic feasibility of BC production processes hinders its industrialization. In our previous work, we obtained candidate strains with improved BC production through random mutations in Gluconacetobacter. In this study, the molecular identification of LYP25 strain with significantly improved productivity, the development of chestnut pericarp (CP) hydrolysate medium, and its application in BC fermentation were performed for cost-effective BC production process. As a result, the mutant strain was identified as Gluconacetobacter xylinus. The CP hydrolysate (CPH) medium contained 30 g/L glucose with 0.4 g/L acetic acid, whereas other candidates known to inhibit fermentation were not detected. Although acetic acid is generally known as a fermentation inhibitor, it improves the BC production by G. xylinus when present within about 5 g/L in the medium. Fermentation of G. xylinus LYP25 in CPH medium resulted in 17.3 g/L BC, a 33 % improvement in production compared to the control medium, and BC from the experimental and control groups had similar physicochemical properties. Finally, the overall process of BC production from biomass was evaluated and our proposed platform showed the highest yield (17.9 g BC/100 g biomass).

Enhanced Enzymatic Synthesis of Puerarin Palmitate with Different Acyl Donors for Lipid Solubility Improvement.

Puerarin is a flavonoid known as a natural antioxidant found in the root of Pueraria robata. Its antioxidant, anticancer, and anti-inflammatory effects have attracted attention as a potential functional ingredient in various bioindustries. However, puerarin has limited bioavailability owing to its low lipid solubility and stability. Acylation is proposed as a synthesis method to overcome this limitation. In this study, lipase-catalyzed acylation of puerarin and various acyl donors was performed, and the enzymatic synthetic condition was optimized. Under the condition (20 g/L of Novozym 435, palmitic anhydride, 1:15, 40 °C, tetrahydrofuran (THF)), the synthesis of puerarin ester achieved a significantly high conversion (98.97%) within a short time (3 h). The molecule of the synthesized puerarin palmitate was identified by various analyses such as liquid chromatography-mass spectrometry (LC-MS), Fourier-transform infrared spectroscopy (FT-IR), and carbon-13 nuclear magnetic resonance (13C NMR). The lipid solubility and the radical scavenging activity were also evaluated. Puerarin palmitate showed a slight decrease in antioxidant activity, but lipid solubility was significantly improved, improving bioavailability. The high conversion achieved for puerarin esters in this study will provide the foundation for industrial applications.

Extended-Gate Field-Effect Transistor Consisted of a CD9 Aptamer and MXene for Exosome Detection in Human Serum.

Cancer progresses silently to the terminal stage of the impossible operable condition. There are many limitations in the treatment options of cancer, but diagnosis in an early stage can improve survival rates and low recurrence. Exosomes are the biomolecules released from cancer cells and are promising candidates for clinical diagnosis. Among them, the cluster of differentiation 9 (CD9) protein is an important exosomal biomarker that can be used for exosome determination. Therefore, here, a CD9 aptamer was first synthesized and applied to an extended-gate field-effect transistor (EGFET)-type biosensor containing a disposable sensing membrane to suggest the possibility of detecting exosomes in a clinical environment. Systematically evaluating ligands using the exponential enrichment (SELEX) technique was performed to select nucleic acid sequences that can specifically target the CD9 protein. Exosomes were detected according to the electrical signal changes on a membrane, which is an extended gate using an Au microelectrode. The fabricated biosensor showed a limit of detection (LOD) of 10.64 pM for CD9 proteins, and the detection range was determined from 10 pM to 1 µM in the buffer. In the case of the clinical test, the LOD and detection ranges of exosomes in human serum samples were 6.41 × 102 exosomes/mL and 1 × 103 to 1 × 107 exosomes/mL, respectively, showing highly reliable results with low error rates. These findings suggest that the proposed aptasensor can be a powerful tool for a simple and early diagnosis of exosomes.

Construction of a rapid electrochemical biosensor consisting of a nanozyme/aptamer conjugate for waterborne microcystin detection.

Microcystin-LR (MC-LR) is a hepatotoxin generated by the excessive proliferation of cyanobacteria, which is a threat to humans and wildlife. Therefore, rapid detection of MC-LR is an important challenge. This study describes a rapid electrochemical biosensor comprising nanozymes and aptamers. Alternating current electrothermal flow (ACEF) significantly reduced the MC-LR detection period to 10 min. We also used MnO2/MC-LR aptamer conjugates to improve the sensitivity to MC-LR detection. Here, MnO2 amplified the electrochemical signal and the aptamer showed high selectivity for MC-LR. Under the optimal conditions, the limit of detection (LOD) and selectivity in freshwater were detected using cyclic voltammetry and differential pulse voltammetry. As a result, an LOD of 3.36 pg mL-1 was observed in the linear concentration range of 10 pg mL-1 to 1 µg mL-1. This study quickly and sensitively detected MC-LR in a situation where it causes serious damage worldwide. In addition, the ACEF technology introduction is the first example of MC-LR detection, suggesting a wide range of possibilities for MC-LR biosensors.

Fabrication of Rapid Electrical Pulse-Based Biosensor Consisting of Truncated DNA Aptamer for Zika Virus Envelope Protein Detection in Clinical Samples.

Zika virus (ZV) infection causes fatal hemorrhagic fever. Most patients are unaware of their symptoms; therefore, a rapid diagnostic tool is required to detect ZV infection. To solve this problem, we developed a rapid electrical biosensor composed of a truncated DNA aptamer immobilized on an interdigitated gold micro-gap electrode and alternating current electrothermal flow (ACEF) technique. The truncated ZV aptamer (T-ZV apt) was prepared to reduce the manufacturing cost for biosensor fabrication, and it showed binding affinity similar to that of the original ZV aptamer. This pulse-voltammetry-based biosensor was composed of a T-ZV apt immobilized on an interdigitated micro-gap electrode. Atomic force microscopy was used to confirm the biosensor fabrication. In addition, the optimal biosensor performance conditions were investigated using pulse voltammetry. ACEF promoted aptamer-target binding, and the target virus envelope protein was detected in the diluted serum within 10 min. The biosensor waveform increased linearly as the concentration of the Zika envelope in the serum increased, and the detection limit was 90.1 pM. Our results suggest that the fabricated biosensor is a significant milestone for rapid virus detection.

Customized valorization of waste streams by Pseudomonas putida: State-of-the-art, challenges, and future trends.

Preventing catastrophic climate events warrants prompt action to delay global warming, which threatens health and food security. In this context, waste management using engineered microbes has emerged as a long-term eco-friendly solution for addressing the global climate crisis and transitioning to clean energy. Notably, Pseudomonas putida can valorize industry-derived synthetic wastes including plastics, oils, food, and agricultural waste into products of interest, and it has been extensively explored for establishing a fully circular bioeconomy through the conversion of waste into bio-based products, including platform chemicals (e.g., cis,cis-muconic and adipic acid) and biopolymers (e.g., medium-chain length polyhydroxyalkanoate). However, the efficiency of waste pretreatment technologies, capability of microbial cell factories, and practicability of synthetic biology tools remain low, posing a challenge to the industrial application of P. putida. The present review discusses the state-of-the-art, challenges, and future prospects for divergent biosynthesis of versatile products from waste-derived feedstocks using P. putida.

Improved production of bacterial cellulose using Gluconacetobacter sp. LYP25, a strain developed in UVC mutagenesis with limited viability conditions.

Bacterial cellulose (BC), a natural polymer synthesized by bacteria, has received considerable attention owing to its impressive physicomechanical properties. However, the low productivity of BC-producing strains poses a challenge to industrializing this material and making it economically viable. In the present study, UV-induced random mutagenesis of Gluconacetobacter xylinus ATCC 53524 was performed to improve BC production. Sixty mutants were obtained from the following mutagenesis procedure: the correlation between UVC fluence and cell death was investigated, and a limited viability condition was determined as a UVC dose to kill 99.99 %. Compared to the control strain, BC production by the mutant strains LYP25 and LYP23 improved 46.4 % and 44.9 %, respectively. Fermentation profiling using the selected strains showed that LYP25 was superior in glucose consumption and BC production, 13.8 % and 41.0 %, respectively, compared to the control strain. Finally, the physicochemical properties of LYP25-derived BC were similar to those of the control strain; thus, the mutant strain is expected to be a promising producer of BC in the bio-industry based on improved productivity.

Green and Sustainable Hot Melt Adhesive (HMA) Based on Polyhydroxyalkanoate (PHA) and Silanized Cellulose Nanofibers (SCNFs).

Polyhydroxyalkanoate (PHA), with a long chain length and high poly(4-hydroxybutyric acid) (P4HB) ratio, can be used as a base polymer for eco-friendly and biodegradable adhesives owing to its high elasticity, elongation at break, flexibility, and processability; however, its molecular structures must be adjusted for adhesive applications. In this study, surface-modified cellulose nanofibers (CNFs) were used as a hydrophobic additive for the PHA-based adhesive. For the surface modification of CNFs, double silanization using tetraethyl orthosilicate (TEOS) and methyltrimethoxysilane (MTMS) was performed, and the thermal and structural properties were evaluated. The hydrophobicity of the TEOS- and MTMS-treated CNFs (TMCNFs) was confirmed by FT-IR and water contact angle analysis, with hydrophobic CNFs well dispersed in the PHA. The PHA-CNFs composite was prepared with TMCNFs, and its morphological analysis verified the good dispersion of TMCNFs in the PHA. The tensile strength of the composite was enhanced when 10% TMCNFs were added; however, the viscosity decreased as the TMCNFs acted as a thixotropic agent. Adding TMCNFs to PHA enhanced the flowability and infiltration ability of the PHA-TMCNFs-based adhesive, and an increase in the loss tangent (Tan δ) and adjustment of viscosity without reducing the adhesive strength was also observed. These changes in properties can improve the flowability and dispersibility of the PHA-TMCNFs adhesive on a rough adhesive surface at low stress. Thus, it is expected that double-silanized CNFs effectively improve their interfacial adhesion in PHA and the adhesive properties of the PHA-CNFs composites, which can be utilized for more suitable adhesive applications.

Microbial Production of Bacterial Cellulose Using Chestnut Shell Hydrolysates by Gluconacetobacter xylinus ATCC 53524.

Bacterial cellulose (BC) is gaining attention as a carbon-neutral alternative to plant cellulose, and as a means to prevent deforestation and achieve a carbon-neutral society. However, the high cost of fermentation media for BC production is a barrier to its industrialization. In this study, chestnut shell (CS) hydrolysates were used as a carbon source for the BC-producing bacteria strain, Gluconacetobacter xylinus ATCC 53524. To evaluate the suitability of the CS hydrolysates, major inhibitors in the hydrolysates were analyzed, and BC production was profiled during fermentation. CS hydrolysates (40 g glucose/l) contained 1.9 g/l acetic acid when applied directly to the main medium. As a result, the BC concentration at 96 h using the control group and CS hydrolysates was 12.5 g/l and 16.7 g/l, respectively (1.3-fold improved). In addition, the surface morphology of BC derived from CS hydrolysates revealed more densely packed nanofibrils than the control group. In the microbial BC production using CS, the hydrolysate had no inhibitory effect during fermentation, suggesting it is a suitable feedstock for a sustainable and eco-friendly biorefinery. To the best of our knowledge, this is the first study to valorize CS by utilizing it in BC production.

A colorimetric detection of polystyrene nanoplastics with gold nanoparticles in the aqueous phase.

Nanoplastics has become a growing environmental concern because these tiny particles can easily penetrate biological tissues and have possible toxic effects. FT-IR and Raman spectroscopy methods are currently used to analyze microplastics (5 mm or less), but they encounter difficulty when the particles are below 1 µm. On the other hand, thermoanalysis of nanoplastics does not provide the size information. Therefore, herein, we proposed the colorimetric detection method with gold nanoparticles (AuNPs) to measure the concentration of polystyrene nanoplastics (PSNPs, size = 350 and 880 nm) through a color change that is obvious to the naked eye. A mixed dispersion of PSNPs and AuNPs was added with acetone, a good solvent for polystyrene. In this colorimetric detection, acetone acted as a key component to enhance or hinder the aggregation of AuNPs around PSNPs. Namely, acetone itself promoted AuNPs aggregation, but it was dissolved partially PS chains from PSNSs to act as inhibitor of aggregation of AuNPs. These two contradictory feature of acetone to AuNPs and PSNPs affect the aggregation and color of AuNPs (dispersed = red, aggregated = blue). Finally, the heat-map between PSNSs and acetone was prepared to use for estimation of concentration of PSNPs in the aqueous solution with naked eye. To the best of our knowledge, this is the first attempt to measure nanoplastics by colorimetry.

Introduction of Nanomaterials to Biosensors for Exosome Detection: Case Study for Cancer Analysis.

Exosomes have been gaining attention for early cancer diagnosis owing to their biological functions in cells. Several studies have reported the relevance of exosomes in various diseases, including pancreatic cancer, retroperitoneal fibrosis, obesity, neurodegenerative diseases, and atherosclerosis. Particularly, exosomes are regarded as biomarkers for cancer diagnosis and can be detected in biofluids, such as saliva, urine, peritoneal fluid, and blood. Thus, exosomes are advantageous for cancer liquid biopsies as they overcome the current limitations of cancer tissue biopsies. Several studies have reported methods for exosome isolation, and analysis for cancer diagnosis. However, further clinical trials are still required to determine accurate exosome concentration quantification methods. Recently, various biosensors have been developed to detect exosomal biomarkers, including tumor-derived exosomes, nucleic acids, and proteins. Among these, the exact quantification of tumor-derived exosomes is a serious obstacle to the clinical use of liquid biopsies. Precise detection of exosome concentration is difficult because it requires clinical sample pretreatment. To solve this problem, the use of the nanobiohybrid material-based biosensor provides improved sensitivity and selectivity. The present review will discuss recent progress in exosome biosensors consisting of nanomaterials and biomaterial hybrids for electrochemical, electrical, and optical-based biosensors.

Development of GO/Co/Chitosan-Based Nano-Biosensor for Real-Time Detection of D-Glucose.

Electrochemical nano-biosensor systems are popular in the industrial field, along with evaluations of medical, agricultural, environmental and sports analysis, because they can simultaneously perform qualitative and quantitative analyses with high sensitivity. However, real-time detection using an electrochemical nano-biosensor is greatly affected by the surrounding environment with the performance of the electron transport materials. Therefore, many researchers are trying to find good factors for real-time detection. In this work, it was found that a composite composed of graphite oxide/cobalt/chitosan had strong stability and electron transfer capability and was applied to a bioelectrochemical nano-biosensor with high sensitivity and stability. As a mediator-modified electrode, the GO/Co/chitosan composite was electrically deposited onto an Au film electrode by covalent boding, while glucose oxidase as a receptor was immobilized on the end of the GO/Co/chitosan composite. It was confirmed that the electron transfer ability of the GO/Co/chitosan composite was excellent, as shown with power density analysis. In addition, the real-time detection of D-glucose could be successfully performed by the developed nano-biosensor with a high range of detected concentrations from 1.0 to 15.0 mM. Furthermore, the slope value composed of the current, per the concentration of D-glucose as a detection response, was significantly maintained even after 14 days.

Fabrication of Large-Area Mullite-Cordierite Composite Substrates for Semiconductor Probe Cards and Enhancement of Their Reliability.

This work aims to fabricate a large-area ceramic substrate for the application of probe cards. Mullite (M) and cordierite (C), which both have a low thermal expansion coefficient, excellent resistance to thermal shock, and high durability, were selected as starting powders. The mullite-cordierite composites were produced through different composition ratios of starting powders (M:C = 100:0, M:C = 90:10, M:C = 70:30, M:C = 50:50, M:C = 30:70, and M:C = 0:100). The effects of composition ratio and sintering temperature on the density, porosity, thermal expansion coefficient, and flexural strength of the mullite-cordierite composite pellets were investigated. The results showed that the mullite-cordierite composite pellet containing 70 wt% mullite and 30 wt% cordierite sintered at 1350 °C performed exceptionally well. Based on these findings, a large-area mullite-cordierite composite substrate with a diameter of 320 mm for use in semiconductor probe cards was successfully fabricated. Additionally, the changes in sheet resistance and flexural strength were measured to determine the effect of the environmental tests on the large-area substrate such as damp heat and thermal shock. The results indicated that the mullite-cordierite composite substrate was extremely reliable and durable.

Novel measurement method of determining PS nanoplastic concentration via AuNPs aggregation with NaCl.

Microplastics, or nanoplastics fragmented to sizes in the nanoscale, can easily penetrate living organisms as well as human organs, increasing the risk of toxicity. However, it is challenging to obtain the size of nanoplastics using thermal analysis methods such as pyrolysis gas chromatography/mass spectrometry or thermal desorption-gas chromatography/mass spectrometry, which are used to analyze nanoplastics. In this study, the coupling effect due to the aggregation of gold nanoparticles (AuNPs) was used to measure the concentration of polystyrene nanoplastics (PSNPs). Experiments were conducted to measure the concentration of PSNPs using an ultraviolet-visible spectrophotometer using the phenomenon that the color of the colloid changes when AuNPs are aggregated. The differences in absorbance before and after aggregation after the addition of NaCl were measured. As a result of the experiment, when 20 mM NaCl was added to the solution in which AuNPs and PSNPs were dispersed, the difference in absorbance before and after aggregation and the concentration of PSNPs exhibited high linearity. In addition, 350 and 880 nm-sized PSNPs could be distinguished from each other because of their different linearities. The concentration of PSNPs was measured easily and conveniently without requiring a skilled operator, expensive analytical equipment; additionally, the process was not time or labor intensive, and it was shown that particle size can be measured by distinguishing particles of different sizes. Electronic Supplementary Material: Supplementary material is available for this article at 10.1007/s11814-022-1153-9 and is accessible for authorized users.

Improved Productivity of Astaxanthin from Photosensitive Haematococcus pluvialis Using Phototaxis Technology.

Haematococcus pluvialis is a microalgae actively studied for the production of natural astaxanthin, which is a powerful antioxidant for human application. However, it is economically disadvantageous for commercialization owing to the low productivity of astaxanthin. This study reports an effective screening strategy using the negative phototaxis of the H. pluvialis to attain the mutants having high astaxanthin production. A polydimethylsiloxane (PDMS)-based microfluidic device irradiated with a specific light was developed to efficiently figure out the phototactic response of H. pluvialis. The partial photosynthesis deficient (PP) mutant (negative control) showed a 0.78-fold decreased cellular response to blue light compared to the wild type, demonstrating the positive relationship between the photosynthetic efficiency and the phototaxis. Based on this relationship, the Haematococcus mutants showing photosensitivity to blue light were selected from the 10,000 random mutant libraries. The M1 strain attained from the phototaxis-based screening showed 1.17-fold improved growth rate and 1.26-fold increases in astaxanthin production (55.12 ± 4.12 mg g-1) in the 100 L photo-bioreactor compared to the wild type. This study provides an effective selection tool for industrial application of the H. pluvialis with improved astaxanthin productivity.

Efficient Production of Naringin Acetate with Different Acyl Donors via Enzymatic Transesterification by Lipases.

Naringin, one of the citrus flavonoids and known as a natural antioxidant, has limited bioavailability owing to its low stability and solubility. However, naringin esters formed via acylation have recently been reported to possess improved physical and chemical properties. The development of these compounds has a great potential in the food, cosmetic and pharmaceutical industries, but low conversion and productivity are barriers to industrial applications. This study aimed to improve the conversion of naringin acetate, which is formed via the enzymatic reaction between naringin and an acyl donor. An optimal reaction condition was determined by evaluating the effect of various variables (enzyme type, enzyme concentration, acyl donor, molar ratio of reactants, reaction temperature, and solvent) on the synthesis of naringin acetate. The optimal condition was as follows: 3 g/L of Lipozyme TL IM, molar ratio of 1:5 (naringin:acyl donor), reaction temperature of 40 °C, and acetonitrile as the reaction solvent. Under this condition, the maximum conversion to naringin acetate from acetic anhydride and vinyl acetate was achieved at approximately 98.5% (8 h) and 97.5% (24 h), respectively. Compared to the previously reported values, a high conversion was achieved within a short time, confirming the commercial potential of the process.

Improved Productivity of Naringin Oleate with Flavonoid and Fatty Acid by Efficient Enzymatic Esterification.

Naringin is a flavonoid found in citrus fruits. It exhibits biological activities, such as anticancer and antioxidant effects, but it suffers from low solubility and low stability in lipophilic systems. These drawbacks lead to difficulties in the commercial application of naringin, but they can be overcome through esterification. In this study, naringin oleate was synthesized by enzymatic esterification and optimal conditions for the reaction were investigated. Experiments were conducted focusing on the following parameters: enzyme type, enzyme concentration, molar ratio of naringin to oleic acid, reaction temperature, and reaction solvent. We further confirmed the degree of esterification based on the difference in the initial and the final naringin concentrations. A conversion of 93.10% was obtained under optimized conditions (Lipozyme TL IM 10 g/L, molar ratio 1:20, reaction temperature 40 °C, acetonitrile as solvent, and 48 h reaction time). Thus, naringin oleate, a high value-added material that overcomes the low hydrophobicity of naringin and enhances its performance, was obtained through esterification of naringin using oleic acid. This study presented a method for the efficient enzymatic synthesis that could ensure high conversion within a shorter reaction time compared with that required in previously reported methods.

Enhanced Production of Bacterial Cellulose from Miscanthus as Sustainable Feedstock through Statistical Optimization of Culture Conditions.

Biorefineries are attracting attention as an alternative to the petroleum industry to reduce carbon emissions and achieve sustainable development. In particular, because forests play an important role in potentially reducing greenhouse gas emissions to net zero, alternatives to cellulose produced by plants are required. Bacterial cellulose (BC) can prevent deforestation and has a high potential for use as a biomaterial in various industries such as food, cosmetics, and pharmaceuticals. This study aimed to improve BC production from lignocellulose, a sustainable feedstock, and to optimize the culture conditions for Gluconacetobacter xylinus using Miscanthus hydrolysates as a medium. The productivity of BC was improved using statistical optimization of the major culture parameters which were as follows: temperature, 29 °C; initial pH, 5.1; and sodium alginate concentration, 0.09% (w/v). The predicted and actual values of BC production in the optimal conditions were 14.07 g/L and 14.88 g/L, respectively, confirming that our prediction model was statistically significant. Additionally, BC production using Miscanthus hydrolysates was 1.12-fold higher than in the control group (commercial glucose). Our result indicate that lignocellulose can be used in the BC production processes in the near future.

Energy-efficient glucose recovery from chestnut shell by optimization of NaOH pretreatment at room temperature and application to bioethanol production.

Biofuel policies are currently being implemented globally to reduce greenhouse gas emissions. The recent European regulation, Renewable Energy Directive (RED) II, states that renewable resources should be used as raw materials. In this study, chestnut shell (CNS), a food processing residue, was utilized as a feedstock for bioethanol production. Statistical optimization was performed to improve biomass-to-glucose conversion (BtG) from the CNS. In order to design an energy-efficient process, the pretreatment was fixed at room temperature in the numerical optimization. The optimal conditions derived from the predicted model are as follows: temperature of 25 °C, reaction time of 2.8 h, and NaOH concentration of 1.9% (w/w). Under optimal conditions, both predicted and experimental BtG were 31.0%, while BtG was approximately 3.3-fold improved compared to the control group (without pretreatment). The recovered glucose was utilized for bioethanol fermentation by Saccharomyces cerevisiae K35 and the ethanol yield was achieved to be 98%. Finally, according to the mass balance based on 1000 g CNS, glucose of 310 g can be recovered by the pretreatment; the bioethanol production was approximately 155 g. This strategy suggests a direction to utilize CNS as a potential feedstock for biorefinery through the design of an economical and energy-efficient pretreatment process by lowering the reaction temperature to room temperature.

Fabrication of MERS-nanovesicle biosensor composed of multi-functional DNA aptamer/graphene-MoS2 nanocomposite based on electrochemical and surface-enhanced Raman spectroscopy.

Middle East respiratory syndrome coronavirus (MERS-CoV) is one of the most harmful viruses for humans in nowadays. To prevent the spread of MERS-CoV, a valid detection method is highly needed. For the first time, a MERS-nanovesicle (NV) biosensor composed of multi-functional DNA aptamer and graphene oxide encapsulated molybdenum disulfide (GO-MoS2) hybrid nanocomposite was fabricated based on electrochemical (EC) and surface-enhanced Raman spectroscopy (SERS) techniques. The MERS-NV aptamer was designed for specifically binding to the spike protein on MERS-NVs and it is prepared using the systematic evolution of ligands by exponential enrichment (SELEX) technique. For constructing a multi-functional MERS aptamer (MF-aptamer), the prepared aptamer was connected to the DNA 3-way junction (3WJ) structure. DNA 3WJ has the three arms that can connect the three individual functional groups including MERS aptamer (bioprobe), methylene blue (signal reporter) and thiol group (linker) Then, GO-MoS2 hybrid nanocomposite was prepared for the substrate of EC/SERS-based MERS-NV biosensor construction. Then, the assembled multifunctional (MF) DNA aptamer was immobilized on GO-MoS2. The proposed biosensor can detect MERS-NVs not only in a phosphate-buffered saline (PBS) solution (SERS LOD: 0.176 pg/ml, EIS LOD: 0.405 pg/ml) but also in diluted 10% saliva (SERS LOD: 0.525 pg/ml, EIS LOD: 0.645 pg/ml).