Evolutionary Design of Self-Templated Supramolecular Fibrils Using M13 Bacteriophage for Tissue Engineering.

Biomaterials in nature form hierarchical structures and functions across various length scales through binding and assembly processes. Inspired by nature, we developed hierarchically organized tissue engineering materials through evolutionary screening and self-templating assembly. Leveraging the M13 bacteriophage (phage), we employed an evolutionary selection process against hydroxyapatite (HA) to isolate HA-binding phage (HAPh). The newly discovered phage exhibits a bimodal length, comprising 950 nm and 240 nm, where the synergistic effect of these dual lengths promotes the formation of supramolecular fibrils with periodic banded structures. The assembled HAPh fibrils show the capability of HA mineralization and the directional growth of osteoblast cells. When applied to a dentin surface, it induces the regeneration of dentin-like tissue structures, showcasing its potential applications as a scaffold in tissue engineering. The integration of evolutionary screening and self-templating assembly holds promise for the future development of hierarchically organized tissue engineering materials.

Truncated M13 phage for smart detection of E. coli under dark field.

BACKGROUND: The urgent need for affordable and rapid detection methodologies for foodborne pathogens, particularly Escherichia coli (E. coli), highlights the importance of developing efficient and widely accessible diagnostic systems. Dark field microscopy, although effective, requires specific isolation of the target bacteria which can be hindered by the high cost of producing specialized antibodies. Alternatively, M13 bacteriophage, which naturally targets E. coli, offers a cost-efficient option with well-established techniques for its display and modification. Nevertheless, its filamentous structure with a large length-diameter ratio contributes to nonspecific binding and low separation efficiency, posing significant challenges. Consequently, refining M13 phage methodologies and their integration with advanced microscopy techniques stands as a critical pathway to improve detection specificity and efficiency in food safety diagnostics. METHODS: We employed a dual-plasmid strategy to generate a truncated M13 phage (tM13). This engineered tM13 incorporates two key genetic modifications: a partial mutation at the N-terminus of pIII and biotinylation at the hydrophobic end of pVIII. These alterations enable efficient attachment of tM13 to diverse E. coli strains, facilitating rapid magnetic separation. For detection, we additionally implemented a convolutional neural network (CNN)-based algorithm for precise identification and quantification of bacterial cells using dark field microscopy. RESULTS: The results obtained from spike-in and clinical sample analyses demonstrated the accuracy, high sensitivity (with a detection limit of 10 CFU/µL), and time-saving nature (30 min) of our tM13-based immunomagnetic enrichment approach combined with AI-enabled analytics, thereby supporting its potential to facilitate the identification of diverse E. coli strains in complex samples. CONCLUSION: The study established a rapid and accurate detection strategy for E. coli utilizing truncated M13 phages as capture probes, along with a dark field microscopy detection platform that integrates an image processing model and convolutional neural network.

Engineered Living Bacteriophage-Enabled Self-Adjuvanting Hydrogel for Remodeling Tumor Microenvironment and Cancer Therapy.

Due to the complexity and heterogeneity in the tumor microenvironment, the efficacy of breast cancer treatment has been significantly impeded. Here, we established a living system using an engineered M13 bacteriophage through chemical cross-linking and biomineralization to remodel the tumor microenvironment. Chemically cross-linking of the engineered bacteriophage gel (M13 Gel) could in situ synthesize photothermal palladium nanoparticles (PdNPs) on the pVIII capsid protein to obtain M13@Pd Gel. In addition, NLG919 was further loaded into a gel to form (M13@Pd/NLG gel) for down-regulating the expression of tryptophan metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1). Both in vitro and in vivo studies showed that the M13 bacteriophage served not only as a cargo-loaded device but also as a self-immune adjuvant, which induced the immunogenic death of tumor cells effectively and down-regulated IDO1 expression. Such a bioactive gel system constructed by natural living materials could reverse immunosuppression and significantly improve the anti-breast cancer response.

Effects of the Magnetic Orientation of M13 Bacteriophage on Phage Display Selection.

Although phage display selection using a library of M13 bacteriophage has become a powerful tool for finding peptides that bind to target materials on demand, a remaining concern of this method is the interference by the M13 main body, which is a huge filament >103  times larger than the displayed peptide, and therefore would nonspecifically adhere to the target or sterically inhibit the binding of the displayed peptide. Meanwhile, filamentous phages are known to be orientable by an external magnetic field. If M13 filaments are magnetically oriented during the library selection, their angular arrangement relative to the target surface would be changed, being expected to control the interference by the M13 main body. This study reports that the magnetic orientation of M13 filaments vertical to the target surface significantly affects the selection. When the target surface was affinitive to the M13 main body, this orientation notably suppressed the nonspecific adhesion. Furthermore, when the target surface was less affinitive to the M13 main body and intrinsically free from the nonspecific adhesion, this orientation drastically changed the population of M13 clones obtained through library selection. The method of using no chemicals but only a physical stimulus is simple, clean, and expected to expand the scope of phage display selection.

Understanding the Role of M13 Bacteriophage Thin Films on a Metallic Nanostructure through a Standard and Dynamic Model.

The dynamic and surface manipulation of the M13 bacteriophage via the meeting application demands the creation of a pathway to design efficient applications with high selectivity and responsivity rates. Here, we report the role of the M13 bacteriophage thin film layer that is deposited on an optical nanostructure involving gold nanoparticles/SiO2/Si, as well as its influence on optical and geometrical properties. The thickness of the M13 bacteriophage layer was controlled by varying either the concentration or humidity exposure levels, and optical studies were conducted. We designed a standard and dynamic model based upon three-dimensional finite-difference time-domain (3D FDTD) simulations that distinguished the respective necessity of each model under variable conditions. As seen in the experiments, the origin of respective peak wavelength positions was addressed in detail with the help of simulations. The importance of the dynamic model was noted when humidity-based experiments were conducted. Upon introducing varied humidity levels, the dynamic model predicted changes in plasmonic properties as a function of changes in NP positioning, gap size, and effective index (this approach agreed with the experiments and simulated results). We believe that this work will provide fundamental insight into understanding and interpreting the geometrical and optical properties of the nanostructures that involve the M13 bacteriophage. By combining such significant plasmonic properties with the numerous benefits of M13 bacteriophage (like low-cost fabrication, multi-wavelength optical characteristics devised from a single structure, reproducibility, reversible characteristics, and surface modification to suit application requirements), it is possible to develop highly efficient integrated plasmonic biomaterial-based sensor nanostructures.

Fabrication of Chiral M13 Bacteriophage Film by Evaporation-Induced Self-Assembly.

Biomacromolecules are likely to undergo self-assembly and show specific collective behavior concentrated in the medium. Although the assembly procedures have been studied for unraveling their mysteries, there are few cases to directly demonstrate the collective behavior and phase transition process in dynamic systems. In the contribution, the drying process of M13 droplet is investigated, and can be successfully simulated by a doctor blade coating method. The morphologies in the deposited film are measured by atomic force microscopy and the liquid crystal phase development is captured in real time using polarized optical microscope. Collective behaviors near the contact line are characterized by the shape of meniscus curve and particle movement velocity. With considering rheological properties and flow, the resultant chiral film is used to align gold nanorods, and this approach can suggest a way to use M13 bacteriophage as a scaffold for the multi-functional chiral structures.

Vertical Self-Assembly of Polarized Phage Nanostructure for Energy Harvesting.

Controlling the shape, geometry, density, and orientation of nanomaterials is critical to fabricate functional devices. However, there is limited control over the morphological and directional characteristics of presynthesized nanomaterials, which makes them unsuitable for developing devices for practical applications. Here, we address this challenge by demonstrating vertically aligned and polarized piezoelectric nanostructures from presynthesized biological piezoelectric nanofibers, M13 phage, with control over the orientation, polarization direction, microstructure morphology, and density using genetic engineering and template-assisted self-assembly process. The resulting vertically ordered structures exhibit strong unidirectional polarization with three times higher piezoelectric constant values than that of in-plane aligned structures, supported by second harmonic generation and piezoelectric force microscopy measurements. The resulting vertically self-assembled phage-based piezoelectric energy harvester (PEH) produces up to 2.8 V of potential, 120 nA of current, and 236 nW of power upon 17 N of force. In addition, five phage-based PEH integrated devices produce an output voltage of 12 V and an output current of 300 nA, simply by pressing with a finger. The resulting device can operate light-emitting diode backlights on a liquid crystal display. Our approach will be useful for assembling many other presynthesized nanomaterials into high-performance devices for various applications.

Exhaustive Search of the Receptor Ligands by the CyCLOPS (Cytometry Cell-Labeling Operable Phage Screening) Technique.

Effective and versatile screening of the peptide ligands capable of selectively binding to diverse receptors is in high demand for the state-of-the-art technologies in life sciences, including probing of specificity of the cell surface receptors and drug development. Complex microenvironment and structure of the surface receptors significantly reduce the possibility to determine their specificity, especially when in vitro conditions are utilized. Previously, we designed a publicly available platform for the ultra-high-throughput screening (uHTS) of the specificity of surface-exposed receptors of the living eukaryotic cells, which was done by consolidating the phage display and flow cytometry techniques. Here, we significantly improved this methodology and designed the fADL-1e-based phage vectors that do not require a helper hyperphage for the virion assembly. The enhanced screening procedure was tested on soluble human leukocyte antigen (HLA) class II molecules and transgenic antigen-specific B cells that express recombinant lymphoid B-cell receptor (BCR). Our data suggest that the improved vector system may be successfully used for the comprehensive search of the receptor ligands in either cell-based or surface-immobilized assays.

Virus-Templated Nickel Phosphide Nanofoams as Additive-Free, Thin-Film Li-Ion Microbattery Anodes.

Transition metal phosphides are a new class of materials generating interest as alternative negative electrodes in lithium-ion batteries. However, metal phosphide syntheses remain underdeveloped in terms of simultaneous control over phase composition and 3D nanostructure. Herein, M13 bacteriophage is employed as a biological scaffold to develop 3D nickel phosphide nanofoams with control over a range of phase compositions and structural elements. Virus-templated Ni5 P4 nanofoams are then integrated as thin-film negative electrodes in lithium-ion microbatteries, demonstrating a discharge capacity of 677 mAh g-1 (677 mAh cm-3 ) and an 80% capacity retention over more than 100 cycles. This strong electrochemical performance is attributed to the virus-templated, nanostructured morphology, which remains electronically conductive throughout cycling, thereby sidestepping the need for conductive additives. When accounting for the mass of additional binder materials, virus-templated Ni5 P4 nanofoams demonstrate the highest practical capacity reported thus far for Ni5 P4 electrodes. Looking forward, this synthesis method is generalizable and can enable precise control over the 3D nanostructure and phase composition in other metal phosphides, such as cobalt and copper.

Mass spectrometry enumeration of filamentous M13 bacteriophage.

In the last decade, filamentous M13 bacteriophage has emerged into numerous biotechnological applications as a promising nontoxic and self-assembling biomaterial with specific binding properties. This raises a question about its upscale production that consequently requires an accurate phage enumeration during the various protocol developments. However, traditional methods of measuring phage concentration are mainly biological in nature and therefore time and labor intensive. These traditional methods also demonstrate poor reproducibility and are semi-quantitative at best. In the present work, we capitalized on mass spectrometry based absolute protein quantitation. We have optimized the quantitation conditions for a major coat protein, pVIII. Enumeration of M13 bacteriophage can be further performed using the determined molar concentration of pVIII, Avogadro's number, and known copy number of pVIII per phage. Since many different phages have well-defined copy number of capsid proteins, the proposed approach can be simply applied to any phage with known copy number of a specific capsid protein.

Deep, noninvasive imaging and surgical guidance of submillimeter tumors using targeted M13-stabilized single-walled carbon nanotubes.

Highly sensitive detection of small, deep tumors for early diagnosis and surgical interventions remains a challenge for conventional imaging modalities. Second-window near-infrared light (NIR2, 950-1,400 nm) is promising for in vivo fluorescence imaging due to deep tissue penetration and low tissue autofluorescence. With their intrinsic fluorescence in the NIR2 regime and lack of photobleaching, single-walled carbon nanotubes (SWNTs) are potentially attractive contrast agents to detect tumors. Here, targeted M13 virus-stabilized SWNTs are used to visualize deep, disseminated tumors in vivo. This targeted nanoprobe, which uses M13 to stably display both tumor-targeting peptides and an SWNT imaging probe, demonstrates excellent tumor-to-background uptake and exhibits higher signal-to-noise performance compared with visible and near-infrared (NIR1) dyes for delineating tumor nodules. Detection and excision of tumors by a gynecological surgeon improved with SWNT image guidance and led to the identification of submillimeter tumors. Collectively, these findings demonstrate the promise of targeted SWNT nanoprobes for noninvasive disease monitoring and guided surgery.

Nanotentacle-structured magnetic particles for efficient capture of circulating tumor cells.

Circulating tumor cells (CTCs) have attracted considerable attention as promising markers for diagnosing and monitoring the cancer status. Despite many technological advances in isolating CTCs, the capture efficiency and purity still remain challenges that limit clinical practice. Here, the construction of "nanotentacle"-structured magnetic particles using M13-bacteriophage and their application for the efficient capturing of CTCs is demonstrated. The M13-bacteriophage to magnetic particles followed by modification with PEG is conjugated, and further tethered monoclonal antibodies against the epidermal receptor 2 (HER2). The use of nanotentacle-structured magnetic particles results in a high capture purity (>45%) and efficiency (>90%), even for a smaller number of cancer cells (≈25 cells) in whole blood. Furthermore, the cancer cells captured are shown to maintain a viability of greater than 84%. The approach can be effectively used for capturing CTCs with high efficiency and purity for the diagnosis and monitoring of cancer status.

Conductivity optimisation of graphene oxide-M13 bacteriophage nanocomposites: towards graphene-based gas micronano-sensors.

Graphene oxide (GO) and M13 bacteriophage can self-assemble to form ultra-low density porous structures, known as GraPhage13 aerogels (GPA). Due to the insulating nature of GPA and the challenges in producing highly conductive aerogels, it is paramount to explore ways to enhance the conductivity of GPA. Herein, we have developed a method to enhance the conductivity of GPA, via the integration and optimisation of 5 nm and 20 nm diameter gold nanoparticles (AuNPs) into the aerogel structure and systematically analysed the morphology, composition and spectroscopic properties of the resulting GPA-Au nanocomposite. The fabricated GPA-Au nanocomposites exhibited remarkable increases in conductivity, with the integration of 5 nm AuNPs leading to a 53-fold increase compared to GPA, achieving a performance of up to 360 nS/cm, which is within the range suitable for miniaturised semiconductor devices. The mechanism behind the conductivity enhancement was further investigated and attributed to GO-AuNP interactions increasing the carrier density by introducing new energy levels in the GO band gap or shifting its Fermi level towards the conduction band. These findings demonstrate the potential of functionalised AuNPs to significantly improve the electrical properties of GPA, paving the way for their application in gas sensors for biological and chemical detection and a new range of advanced semiconductor devices.

Multichannel Hierarchical Analysis of Time-Resolved Hyperspectral Data for Advanced Colorimetric E-Nose.

The colorimetric sensor-based electronic nose has been demonstrated to discriminate specific gaseous molecules for various applications, including health or environmental monitoring. However, conventional colorimetric sensor systems rely on RGB sensors, which cannot capture the complete spectral response of the system. This limitation can degrade the performance of machine learning analysis, leading to inaccurate identification of chemicals with similar functional groups. Here, we propose a novel time-resolved hyperspectral (TRH) data set from colorimetric array sensors consisting of 1D spatial, 1D spectral, and 1D temporal axes, which enables hierarchical analysis of multichannel 2D spectrograms via a convolution neural network (CNN). We assessed the outstanding classification performance of the TRH data set compared to an RGB data set by conducting a relative humidity (RH) concentration classification. The time-dependent spectral response of the colorimetric sensor was measured and trained as a CNN model using TRH and RGB sensor systems at different RH levels. While the TRH model shows a high classification accuracy of 97.5% for the RH concentration, the RGB model yields 72.5% under identical conditions. Furthermore, we demonstrated the detection of various functional volatile gases with the TRH system by using experimental and simulation approaches. The results reveal distinct spectral features from the TRH system, corresponding to changes in the concentration of each substance.

Helical Hybrid Nanostructure Based on Chiral M13 Bacteriophage via Evaporation-Induced Three-Dimensional Process.

The use of naturally sourced organic materials with chirality, such as the M13 bacteriophage, holds intriguing implications, especially in the field of nanotechnology. The chirality properties of bacteriophages have been demonstrated through numerous studies, particularly in the analysis of liquid crystal phase transitions, developing specific applications. However, exploring the utilization of the M13 bacteriophage as a template for creating chiral nanostructures for optics and sensor applications comes with significant challenges. In this study, the chirality of the M13 bacteriophage was leveraged as a valuable tool for generating helical hybrid structures by combining it with nanoparticles through an evaporation-induced three-dimensional (3D) printing process. Utilizing on the self-assembly property of the M13 bacteriophage, metal nanoparticles were organized into a helical chain under the influence of the M13 bacteriophage at the meniscus interface. External parameters, including nanoparticle shape, the ratio between the bacteriophage and nanoparticles, and pulling speed, were demonstrated as crucial factors affecting the fabrication of helical nanostructures. This study aimed to explore the potential of chiral nanostructure fabrication by utilizing the chirality of the M13 bacteriophage and manipulating external parameters to control the properties of the resulting hybrid structures.

Detection of Micrometer-Sized Virus Aerosols by Using a Real-Time Bioaerosol Monitoring System.

This study investigates a real-time handheld bioaerosol monitoring system for the detection of biological particles using UV-LED and light-induced fluorescence technology. Biological particles produce both scattering and fluorescence signals simultaneously, which can help distinguish them from general particles. The detected scattering, fluorescence, and simultaneous signals are then converted into photon signals and categorized based on predetermined criteria. A reliable biological particle generator was required to validate the performance of the system. This study explores the use of an M13 bacteriophage as a virus simulant of biological agents and employs a customized inkjet aerosol generator to produce M13 bacteriophage aerosols of a specific size by controlling the concentration of M13. We confirmed that micro-sized, narrowly dispersed M13 aerosols were efficiently generated. Additionally, we confirmed the performance of this real-time handheld bioaerosol monitoring system by detecting viruses.

Non-intrusive quality appraisal of differentiation-induced cardiovascular stem cells using E-Nose sensor technology.

Stem cell technology holds immense potential for revolutionizing medicine, particularly in regenerative treatment for heart disease. The unique capacity of stem cells to differentiate into diverse cell types offers promise in repairing damaged tissues and implanting organs. Ensuring the quality of differentiated cells, essential for specific functions, demands in-depth analysis. However, this process consumes time and incurs substantial costs while invasive methods may alter stem cell features during differentiation and deplete cell numbers. To address these challenges, we propose a non-invasive strategy, using cellular respiration, to assess the quality of differentiation-induced stem cells, notably cardiovascular stem cells. This evaluation employs an electronic nose (E-Nose) and neural pattern separation (NPS). Our goal is to assess differentiation-induced cardiac stem cells (DICs) quality through E-Nose data analysis and compare it with standard commercial human cells (SCHCs). Sensitivity and specificity were evaluated by interacting SCHCs and DICs with the E-Nose, achieving over 90% classification accuracy. Employing selective combinations optimized by NPS, E-Nose successfully classified all six cell types. Consequently, the relative similarity among DICs like cardiomyocytes, endothelial cells with SCHCs was established relied on comparing response data from the E-Nose sensor without resorting to complex evaluations.

Combining a Base Deaminase Mutator with Phage-Assisted Evolution.

Phage-assisted evolution has emerged as a powerful technique for improving a protein's function by using mutagenesis and selective pressure. However, mutations typically occur throughout the host's genome and are not limited to the gene-of-interest (GOI): these undesirable genomic mutations can yield host cells that circumvent the system's selective pressure. Our system targets mutations specifically toward the GOI by combining T7 targeted mutagenesis and phage-assisted evolution. This system improves the structure and function of proteins by accumulating favorable mutations that can change its binding affinity, specificity, and activity.

M13KO7 bacteriophage enables Potato Virus Y detection.

IMPORTANCE: In this study, we confirmed the binding of M13KO7 to Potato virus Y (PVY) using enzyme-linked immunosorbent assay. M13KO7 is a "bald" bacteriophage in which no recombinant antibody is displayed. M13KO7 is easy to propagate by using Escherichia coli, making this method more reasonable in economic perspective. Based on this study, we suggest that M13KO7 detection system has applicability as a novel biological tool for the detection of PVY.

Artificial Neural Processing-Driven Bioelectronic Nose for the Diagnosis of Diabetes and Its Complications.

Diabetes and its complications affect the younger population and are associated with a high mortality rate; however, early diagnosis can contribute to the selection of appropriate treatment regimens that can reduce mortality. Although diabetes diagnosis via exhaled breath has great potential for early diagnosis, research on such diagnosis is restricted to disease detection, requiring in-depth examination to diagnose and classify diseases and their complications. This study demonstrates the use of an artificial neural processing-based bioelectronic nose to accurately diagnose diabetes and classify diabetic types (type I and II) and their complications, such as heart disease. Specifically, an M13 phage-based electronic nose (e-nose) is used to explore the features of subjects with diabetes at various levels of cellular and organismal organization (cells, liver organoids, and mice). Exhaled breath samples are collected during culturing and exposed to the phage-based e-nose. Compared with cells, liver organoids cultured under conditions mimicking a diabetic environment display properties that closely resemble the characteristics of diabetic mice. Using neural pattern separation, the M13 phage-based e-nose achieves a classification success rate of over 86% for four conditions in mice, namely, type 1 diabetes, type 2 diabetes, diabetic cardiomyopathy, and cardiomyopathy.