Nanotopology-Enabled On-Site Pathogen Detection for Managing Atopic Dermatitis.

Atopic dermatitis (AD), a prevalent skin condition often complicated by microbial infection, poses a significant challenge in identifying the responsible pathogen for its effective management. However, a reliable, safe tool for pinpointing the source of these infections remains elusive. In this study, a novel on-site pathogen detection that combines chemically functionalized nanotopology with genetic analysis is proposed to capture and analyze pathogens closely associated with severe atopic dermatitis. The chemically functionalized nanotopology features a 3D hierarchical nanopillar array (HNA) with a functional polymer coating, tailored to isolate target pathogens from infected skin. This innovative nanotopology demonstrates superior pathogenic capture efficiency, favorable entrapment patterns, and non-cytotoxicity. An HNA-assembled stick is utilized to directly retrieve bacteria from infected skin samples, followed by extraction-free quantitative loop-mediated isothermal amplification (direct qLAMP) for validation. To mimic human skin conditions, porcine skin is employed to successfully capture Staphylococcus aureus, a common bacterium exacerbating AD cases. The on-site detection method exhibits an impressive detection limit of 103 cells mL-1 . The HNA-assembled stick represents a promising tool for on-site detection of bacteria associated with atopic dermatitis. This innovative approach enables to deepen the understanding of AD pathogenesis and open avenues for more effective management strategies for chronic skin conditions.

Streamlined Specimen Purification for Rapid COVID-19 Diagnosis Using Positively Charged Polymer Thin Film-Coated Surfaces and Chamber Digital PCR.

The ongoing coronavirus disease 2019 (COVID-19) pandemic demands rapid and straightforward diagnostic tools to prevent early-stage viral transmission. Although nasopharyngeal swabs are a widely used patient sample collection method for diagnosing COVID-19, using these samples for diagnosis without RNA extraction increases the risk of obtaining false-positive and -negative results. Thus, multiple purification steps are necessary, which are time-consuming, generate significant waste, and result in substantial sample loss. To address these issues, we developed surface-modified polymerase chain reaction (PCR) tubes using the tertiary aminated polymer poly(2-dimethylaminomethylstyrene) (pDMAMS) via initiated chemical vapor deposition. Introducing the clinical samples into the pDMAMS-coated tubes resulted in approximately 100% RNA capture efficiency within 25 min, which occurred through electrostatic interactions between the positively charged pDMAMS surface and the negatively charged RNA. The captured RNA is then detected via chamber digital PCR, enabling a sensitive, accurate, and rapid diagnosis. Our platform provides a simple and efficient RNA extraction and detection strategy that allows detection from 22 nasopharyngeal swabs and 21 saliva specimens with 0% false negatives. The proposed method can facilitate the diagnosis of COVID-19 and contribute to the prevention of early-stage transmission.

Charge-shifting polyplex as a viral RNA extraction carrier for streamlined detection of infectious viruses.

The recent outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for rapid, user-friendly nucleic acid testing that involves simple but efficient RNA extraction. Here, we present a charge-shifting polyplex as an RNA extraction carrier for advanced diagnosis of infectious viral diseases. The polyplex comprises poly(2-(dimethylamino) ethyl acrylate) (pDMAEA) electrostatically conjugated with RNA. The pDMAEA film can rapidly dissolve in the viral RNA solution, promoting immediate binding with RNA to form the polyplex, which enables the efficient capture of a substantial quantity of RNA. Subsequently, the captured RNA can be readily released by the quick hydrolysis of pDMAEA at the onset of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), streamlining the entire process from RNA extraction to analysis. The developed method requires only 5 min of centrifugation and enables the detection of RNA in a one-pot setup. Moreover, the proposed method is fully compatible with high-speed qRT-PCR kits and can identify clinical samples within 1 h including the entire extraction to detection procedure. Indeed, the method successfully detected influenza viruses, SARS-CoV-2, and their delta and omicron variants in 260 clinical samples with a sensitivity of 99.4% and specificity of 98.9%. This rapid, user-friendly polyplex-based approach represents a significant breakthrough in molecular diagnostics.

Polyaniline-based 3D network structure promotes entrapment and detection of drug-resistant bacteria.

Sensitive and accurate capture, enrichment, and identification of drug-resistant bacteria on human skin are important for early-stage diagnosis and treatment of patients. Herein, we constructed a three-dimensional hierarchically structured polyaniline nanoweb (3D HPN) to capture, enrich, and detect drug-resistant bacteria on-site by rubbing infected skins. These unique hierarchical nanostructures enhance bacteria capture efficiency and help severely deform the surface of the bacteria entrapped on them. Therefore, 3D HPN significantly contributes to the effective and reliable recovery of drug-resistant bacteria from the infected skin and the prevention of potential secondary infection. The recovered bacteria were successfully identified by subsequent real-time polymerase chain reaction (PCR) analysis after the lysis process. The molecular analysis results based on a real-time PCR exhibit excellent sensitivity to detecting target bacteria of concentrations ranging from 102 to 107 CFU/mL without any fluorescent signal interruption. To confirm the field applicability of 3D HPN, it was tested with a drug-resistant model consisting of micropig skin similar to human skin and Klebsiella pneumoniae carbapenemase-producing carbapenem-resistant Enterobacteriaceae (KPC-CRE). The results show that the detection sensitivity of this assay is 102 CFU/mL. Therefore, 3D HPN can be extended to on-site pathogen detection systems, along with rapid molecular diagnostics through a simple method, to recover KPC-CRE from the skin.

Elution-free DNA detection using CRISPR/Cas9-mediated light-up aptamer transcription: Toward all-in-one DNA purification and detection tube.

Accurate and efficient detection of DNA is crucial for disease diagnosis and health monitoring. The traditional methods for DNA analysis involve multiple steps, including sample preparation, lysis, extraction, amplification, and detection. In this study, we present a one-step elution-free DNA analysis method based on the combination of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated light-up aptamer transcription (CLAT) assay and a DNA-capturing poly(2-dimethylaminomethyl styrene) (pDMAMS)-coated tube. The sample solution and lysis buffer are added to the pDMAMS-coated tube, and the DNA is efficiently captured on the surface via electrostatic interaction and directly detected by CLAT assay. The ability of the CRISPR/Cas9 system to specifically recognize DNA enables direct detection of DNA captured on the pDMAMS-coated tube. The combination of CLAT assay and pDMAMS-coated tube simplifies DNA detection in a single tube without the need for complicated extraction steps, improving sensitivity. Our platform demonstrated attomolar sensitivity in the detection of target DNA in cell lysate (0.92 aM), urine (7.7 aM), and plasma (94.6 aM) samples within 1 h. The practical applicability of this method was further demonstrated in experiments with tumor-bearing mice. We believe that this approach brings us closer to an all-in-one DNA purification and detection tube system and has potential applications in tissue and liquid biopsies, as well as various other DNA sensing applications.

Touchable 3D hierarchically structured polyaniline nanoweb for capture and detection of pathogenic bacteria.

A bacteria-capturing platform is a critical function of accurate, quantitative, and sensitive identification of bacterial pathogens for potential usage in the detection of foodborne diseases. Despite the development of various nanostructures and their surface chemical modification strategies, relative to the principal physical contact propagation of bacterial infections, mechanically robust and nanostructured platforms that are available to capture bacteria remain a significant problem. Here, a three-dimensional (3D) hierarchically structured polyaniline nanoweb film is developed for the efficient capture of bacterial pathogens by hand-touching. This unique nanostructure ensures sufficient mechanical resistance when exposed to compression and shear forces and facilitates the 3D interfacial interactions between bacterial extracellular organelles and polyaniline surfaces. The bacterial pathogens (Escherichia coli O157:H7, Salmonella enteritidis, and Staphylococcus aureus) are efficiently captured through finger-touching, as verified by the polymerase chain reaction (PCR) analysis. Moreover, the real-time PCR results of finger-touched cells on a 3D nanoweb film show a highly sensitive detection of bacteria, which is similar to those of the real-time PCR using cultured cells without the capturing step without any interfering of fluorescence signal and structural deformation during thermal cycling.

3D Hierarchical Polyaniline-Metal Hybrid Nanopillars: Morphological Control and Its Antibacterial Application.

Effective and reliable antibacterial surfaces are in high demand in modern society. Although recent works have shown excellent antibacterial performance by combining unique hierarchical nanotopological structures with functional polymer coating, determining the antibacterial performance arising from morphological changes is necessary. In this work, three-dimensional (3D) hierarchical polyaniline-gold (PANI/Au) hybrid nanopillars were successfully fabricated via chemical polymerization (i.e., dilute method). The morphology and structures of the PANI/Au nanopillars were controlled by the reaction time (10 min to 60 h) and the molar concentrations of the monomer (0.01, 0.1, and 1 M aniline), oxidant (0.002, 0.0067, 0.01, and 0.02 M ammonium persulfate), and acid (0.01, 0.1, 1, and 2 M perchloric acid). These complex combinations allow controlling the hierarchical micro- to nanostructure of PANI on a nanopillar array (NPA). Furthermore, the surface of the 3D PANI/Au hierarchical nanostructure can be chemically treated while maintaining the structure using initiated chemical vapor deposition. Moreover, the excellent antibacterial performance of the 3D PANI/Au hierarchical nanostructure (HNS) exceeds 99% after functional polymer coating. The excellent antibacterial performance of the obtained 3D PANI/Au HNS is mainly because of the complex topological and physicochemical surface modification. Thus, these 3D PANI/Au hierarchical nanostructures are promising high-performance antibacterial materials.

All-in-One DNA Extraction Tube for Facilitated Real-Time Detection of Infectious Pathogens.

An "all-in-one tube" platform is developed, where the genetic analysis involving DNA extraction, amplification, and detection can be performed in a single tube. The all-in-one tube consists of a polymerase chain reaction (PCR) tube in which the inner surface is conformally modified with a tertiary-amine-containing polymer to generate a strong electrostatic interaction with DNA. The all-in-one tube provides high DNA capture efficiency exceeding 80% from Escherichia coli O157: H7 pathogen at a wide range of DNA amount from 0.003 to 3 ng. Indeed, the use of the surface-functionalized PCR tube enables direct amplification and detection of the surface-captured DNA without the modification of standard real-time PCR instrument. Besides, this platform has sensitivity, selectivity, and reliability enough for accurate detection at the minimal infective dose of both gram-positive and negative pathogens. The all-in-one tube enables the direct molecular diagnosis, substantially reducing the labor-intensive pathogen detection steps while providing high compatibility with the currently established real-time PCR instruments, and illustrates its on-site applicability with convenience expandable to various genetic analyses including food safety testing, forensic analysis, and clinical diagnosis.

Multifunctional Printable Micropattern Array for Digital Nucleic Acid Assay for Microbial Pathogen Detection.

The digital nucleic acid assay is a precise, sensitive, and reproducible method for determining the presence of individual target molecules separated in designated partitions; thus, this technique can be used for the nucleic acid detection. Here, we propose a multifunctional micropattern array capable of isolating individual target molecules into partitions and simultaneous on-site cell lysis to achieve a direct DNA extraction and digitized quantification thereof. The multifunctional micropattern array is fabricated by the deposition of a copolymer film, poly(2-dimethylaminomethyl styrene-co-hydroxyethyl methacrylate) (pDH), directly on a microfluidic chip surface via the photoinitiated chemical vapor deposition process, followed by hydrophobic microcontact printing (µCP) to define each partition for the nucleic acid isolation. The pDH layer is a positively charged surface, which is desirable for the bacterial lysis and DNA capture, while showing exceptional water stability for more than 24 h. The hydrophobic µCP-treated pDH surface is stable under aqueous conditions at a high temperature (70 °C) for 1 h and enables the rapid and reliable formation of thousands of sessile microdroplets for the compartmentalization of an aqueous sample solution without involving bulky and costly microfluidic devices. By assembling the multifunctional micropattern array into the microfluidic chip, the isothermal amplification in each partition can detect DNA templates over a concentration range of 0.01-2 ng/µL. The untreated bacterial cells can also be directly compartmentalized via the microdroplet formation, followed by the on-site cell lysis and DNA capture on the compartmentalized pDH surface. For Escherichia coli O157:H7, Salmonella enteritidis, and Staphylococcus aureus cells, cell numbers ranging from 1.4 × 104 to 1.4 × 107 can be distinguished by using the multifunctional micropattern array, regardless of the cell type. The multifunctional micropattern array developed in this study provides a novel multifunctional compartmentalization method for rapid, simple, and accurate digital nucleic acid assays.

3D Hierarchical Nanotopography for On-Site Rapid Capture and Sensitive Detection of Infectious Microbial Pathogens.

Effective capture and rapid detection of pathogenic bacteria causing pandemic/epidemic diseases is an important task for global surveillance and prevention of human health threats. Here, we present an advanced approach for the on-site capture and detection of pathogenic bacteria through the combination of hierarchical nanostructures and a nuclease-responsive DNA probe. The specially designed hierarchical nanocilia and network structures on the pillar arrays, termed 3D bacterial capturing nanotopographical trap, exhibit excellent mechanical reliability and rapid (<30 s) and irreversible bacterial capturability. Moreover, the nuclease-responsive DNA probe enables the highly sensitive and extremely fast (<1 min) detection of bacteria. The bacterial capturing nanotopographical trap (b-CNT) facilitates the on-site capture and detection of notorious infectious pathogens (Escherichia coli O157:H7, Salmonella enteritidis, Staphylococcus aureus, and Bacillus cereus) from kitchen tools and food samples. Accordingly, the usefulness of the b-CNT is confirmed as a simple, fast, sensitive, portable, and robust on-site capture and detection tool for point-of-care testing.

Antibacterial Nanopillar Array for an Implantable Intraocular Lens.

Postsurgical intraocular lens (IOL) infection caused by pathogenic bacteria can result in blindness and often requires a secondary operation to replace the contaminated lens. The incorporation of an antibacterial property onto the IOL surface can prevent bacterial infection and postoperative endophthalmitis. This study describes a polymeric nanopillar array (NPA) integrated onto an IOL, which captures and eradicates the bacteria by rupturing the bacterial membrane. This is accomplished by changing the behavior of the elastic nanopillars using bending, restoration, and antibacterial surface modification. The combination of the polymer coating and NPA dimensions can decrease the adhesivity of corneal endothelial cells and posterior capsule opacification without causing cytotoxicity. An ionic antibacterial polymer layer is introduced onto an NPA using an initiated chemical vapor deposition process. This improves bacterial membrane rupture efficiency by increasing the interactions between the bacteria and nanopillars and damages the bacterial membrane using quaternary ammonium compounds. The newly developed ionic polymer-coated NPA exceeds 99% antibacterial efficiency against Staphylococcus aureus, which is achieved through topological and physicochemical surface modification. Thus, this paper provides a novel, efficient strategy to prevent postoperative complications related to bacteria contamination of IOL after cataract surgery.

Development of zinc oxide-based sub-micro pillar arrays for on-site capture and DNA detection of foodborne pathogen.

Prevention and early detection of bacterial infection caused by foodborne pathogens are the most important task to human society. Although currently available diagnostic technologies have been developed and designed for detection of specific pathogens, suitable capturing tools for the pathogens are rarely studied. In this paper, a new methodology is developed and proposed to realize effective capturing through touchable flexible zinc oxide-based sub-micro pillar arrays through genetic analysis. Zinc oxide coated pillar arrays have a high surface area, flexible, and adheres strongly to bacteria. Therefore, it contributes to enhance the bacterial capturability. An in-depth analysis on the sub-sequential capturing process at the bacterial cell-pillar interface is presented. By carefully observing the structural changes and performing numerical analysis under different reaction times, the results are presented. The resulting zinc oxide coated pillar arrays exhibited comprehensive capturability. These pillars were able to detect pathogenic bacteria due to a combination of complex structures, depletion force, and high surface electrostatics. The developed sub-micro pillars successfully captured and detected infectious foodborne bacteria of Escherichia coli in the range of 106-101 CFU/mL.

Naked eye detection of an amplified gene using metal particle-based DNA transport within functionalized porous interfaces.

The current study focuses on developing a system for visually detecting an amplified bacterial (Escherichia coli O157:H7) gene using a heavy metal particle (MP) and functionalized porous sepharose gel. To functionalize DNA-specificity to the MP, an avidin-modified MP was employed in combination with a biotin-conjugated primer. The porous sepharose matrix was functionalized with an amine-reactive group, such as N-hydroxysuccinimide (NHS), to achieve separation upon binding of the amplified gene. The pristine avidin-MPs strongly react with NHS-sepharose via imide bonds owing to the exposure of the amine group on the avidin-MP surface. Conversely, together with the amplified gene, the avidin-MPs are relatively less interactive toward the sepharose gel by steric hindrance of the amplified gene toward the imide bond between NHS and the amine groups. Owing to the higher molecular mass of the MP, those metal particles complexed with the amplified gene pass through the sepharose matrix when centrifugal force is applied. The MPs that are thus separated can be easily visualized by the naked eye owing to their inherent reddish-brown color. A polymerase chain reaction (PCR) product of E. coli O157:H7, present in concentrations ranging from 1.0 × 101 to 1.0 × 106 colony forming units (CFU), in actual food sample was evaluated with high sensitivity and reproducibility. We expect that the MP-based sensing system, which allows for visual detection of PCR-amplified genes, can be clinically used as a point-of-care testing device.

A Disposable and Multi-Chamber Film-Based PCR Chip for Detection of Foodborne Pathogen.

Since the increment of the threat to public health caused by foodborne pathogens, researches have been widely studied on developing the miniaturized detection system for the on-site pathogen detection. In the study, we focused on the development of portable, robust, and disposable film-based polymerase chain reaction (PCR) chip containing a multiplex chamber for simultaneous gene amplification. In order to simply fabricate and operate a film-based PCR chip, different kinds of PCR chambers were designed and fabricated using polyethylene terephthalate (PET) and polyvinyl chloride (PVC) adhesive film, in comparison with commercial PCR, which employs a stereotyped system at a bench-top scale. No reagent leakage was confirmed during the PCR thermal cycling using the film PCR chip, which indicates that the film PCR chip is structurally stable for rapid heat cycling for DNA amplification. Owing to use of the thin film to fabricate the PCR chip, we are able to realize fast thermal transfer from the heat block that leads to short PCR amplification time. Moreover, using the film PCR chip, we could even amplify the target pathogen with 10 CFU mL-1. The artificially infected milk with various concentration of Bacillus cereus was successfully amplified on a single film PCR chip. On the basis of the reliable results, the developed film PCR chip could be a useful tool as a POCT device to detect foodborne pathogens via genetic analysis.

Flexible nanopillar-based electrochemical sensors for genetic detection of foodborne pathogens.

Flexible and highly ordered nanopillar arrayed electrodes have brought great interest for many electrochemical applications, especially to the biosensors, because of its unique mechanical and topological properties. Herein, we report an advanced method to fabricate highly ordered nanopillar electrodes produced by soft-/photo-lithography and metal evaporation. The highly ordered nanopillar array exhibited the superior electrochemical and mechanical properties in regard with the wide space to response with electrolytes, enabling the sensitive analysis. As-prepared gold and silver electrodes on nanopillar arrays exhibit great and stable electrochemical performance to detect the amplified gene from foodborne pathogen of Escherichia coli O157:H7. Additionally, lightweight, flexible, and USB-connectable nanopillar-based electrochemical sensor platform improves the connectivity, portability, and sensitivity. Moreover, we successfully confirm the performance of genetic analysis using real food, specially designed intercalator, and amplified gene from foodborne pathogens with high reproducibility (6% standard deviation) and sensitivity (10 × 1.01 CFU) within 25 s based on the square wave voltammetry principle. This study confirmed excellent mechanical and chemical characteristics of nanopillar electrodes have a great and considerable electrochemical activity to apply as genetic biosensor platform in the fields of point-of-care testing (POCT).

Ultrasonic fabrication of flexible antibacterial ZnO nanopillar array film.

Antibacterial activity is essential and highly demanded in worldwide to prevent potential bacterial infection. Here in this work, we report a new approch for the fabrication of flexible zinc oxide nanopillar arrays (ZG-NPA) film with an efficient antibacterial activity. A flexible NPA film served as a substrate for the rapid formation of ZnO by using ultrasound-assisted method. The enhancement of antibacterial activity were induced by cellular damages because of nano topological effects and electrostatic interaction between bacteria and ZG-NPA. Owing to the benefits of combination with flexibility, high surface areas from nano-features and excellent antibacterial efficiency (>80%) of ZG-NPA, the film can show great potential for use as novel biomaterials for preventing bacterial infections.

Preparation of concave magnetoplasmonic core-shell supraparticles of gold-coated iron oxide via ion-reducible layer-by-layer method for surface enhanced Raman scattering.

Preparation of suprastructure assemblies with unique colloidal and optical properties remains challenging. Non-uniform covering of magnetic nanoparticles (NPs) with an external inert Au shell has been attempted to protect the magnetic core against oxidation as well as to produce multifunctional supraparticles (SPs) possessing respective optical and magnetic properties. In this study, a concave Au NP coating was deposited on magnetic nanoparticles (MNPs) with precise control of the shell thickness and roughness through a layer-by-layer (LbL) assisted ionic reduction method termed ion-reducible LbL (IR-LbL) method. Surface enhanced Raman spectra were obtained using graphene quantum dots (GQDs) on the magnetically aligned structure of the prepared core-shell SPs. It is probable that this synthesis method and the generated SPs are essential for characterizing the merge of electronics and magnetism in the nano-regime and may be applicable for further electronics, magnetic storage, and biomedical applications.

In vivo study on the biocompatibility of chitosan-hydroxyapatite film depending on degree of deacetylation.

Chitosan, produced from chitin, is one of the polymers with promising applications in various fields. However, despite diverse research studies conducted on its biocompatibility, its uses are still limited. The main reason is the degree of deacetylation (DOD), which represents the proportion of deacetylated units in the polymer and is directly correlated with its biocompatibility property. In this article, the in vivo biocompatibility of three chitosan-hydroxyapatite composite films composed of chitosan with different DOD values was investigated by traditional biological protocols and novel optical spectroscopic analyses. The DOD of the chitosan obtained from three different manufacturers was estimated and calculated by Raman spectroscopy, Fourier transform infrared spectroscopy, and proton nuclear magnetic resonance spectroscopy. The chitosan with the higher DOD induced a higher incidence of inflammation in skin cells. The amino group density, biodegradability, and crystallinity of chitosan are the three possible factors that need to be considered when determining the biocompatibility of the films for in vivo application, as they led to complicated biological results, resulting in either better or worse inflammation even when using chitosan products with the same DOD. This basic study on the relationship between the DOD and inflammation is valuable for the development of further chitosan-based researches. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1637-1645, 2017.