Binrui Cao

Virus-based chemical and biological sensing

Viruses have recently proven useful for the detection of target analytes such as explosives, proteins, bacteria, viruses, spores, and toxins with high selectivity and sensitivity. Bacteriophages (often shortened to phages), viruses that specifically infect bacteria, are currently the most studied viruses, mainly because target-specific nonlytic phages (and the peptides and proteins carried by them) can be identified by using the well-established phage display technique, and lytic phages can specifically break bacteria to release cell-specific marker molecules such as enzymes that can be assayed. In addition, phages have good chemical and thermal stability, and can be conjugated with nanomaterials and immobilized on a transducer surface in an analytical device. This Review focuses on progress made in the use of phages in chemical and biological sensors in combination with traditional analytical techniques. Recent progress in the use of virus-nanomaterial composites and other viruses in sensing applications is also highlighted.

Nanofibrous bio-inorganic hybrid structures formed through self-assembly and oriented mineralization of genetically engineered phage nanofibers.

Nanofibrous organic–inorganic hybrid structures, where inorganic nanocomponents are formed or assembled within the aligned organic nanofibrous matrix, are important materials that can find applications in electronics, photonics, catalysis, and tissue engineering.[1–11] They not only provide a means for supporting and ordering the functional inorganic materials such as nanoparticles,[4–6,8] but also can serve as building blocks to further self-assemble into higher-order structures.[5,11,12] The common approaches to the synthesis of the nanofibrous organic-inorganic hybrid structures include electrospinning,[2,4,10] polymer-templating,[3] biotemplating,[5–7,11] and directional freezing.[1] As a matter of fact, the nanofibrous organic-inorganic hybrid structures are important building blocks in natural mineralized biomaterials. One of the best examples is the mineralized collagen fibrils constituting the extracellular matrix (ECM) of bone.[13] Bone is made of cells embedded in ECM, which is hierarchically organized from proteins, including type I collagen and non-collagenous proteins (NCPs) such as bone sialoprotein (BSP), and calcium hydroxylapatite (HAP, Ca10(PO4)6(OH)2). The collagen molecules (~1.5 nm wide and 300 nm long) are self-assembled into wider (up to 200 nm wide) and longer (several µm long) fibrils in a side-to-side and head-to-tail format, which are further hierarchically self-assembled to form much wider and longer collagen fibers (up to several tens µm wide and long).[14,15] HAP is found within the gaps and grooves of the collagen fibers with its c-axis preferentially along the collagen fibers.

Stem cells loaded with nanoparticles as a drug carrier for in vivo breast cancer therapy

A novel anti-cancer drug carrier, mesenchymal stem cells (MSCs) encapsulating drug-loaded hollow silica nanoparticles, is used to carry a photosensitizer drug and deliver it to breast tumors, due to the natural high tumor affinity of the MSCs, and inhibit tumor growth by photo dynamic therapy. This new strategy for delivering a photo sensitizer to tumors by using tumor-affinitive MSCs addresses the challenge of the accumulation of photosensitizer drugs in tumors in photodynamic therapy.

Self-Assembly of Drug-Loaded Liposomes on Genetically Engineered Target-Recognizing M13 Phage: A Novel Nanocarrier for Targeted Drug Delivery**

Liposomes have been a center of research for many years due to their numerous applications in chemistry, biology, medicine, and nanotechnology. They can be used to entrap materials such as drugs either within the central aqueous compartment if they are water soluble, or within the hydrophobic domain of the lipid bilayer if they are oil soluble. In addition, their surface can bemodified to realize targeted delivery. For example, in biomedicine, liposomes are used as vehicles to deliver drugs and genes to specific parts of the body. When used in the delivery of certain anticancer drugs, liposomes help to shield healthy cells from the drug toxicity and prevent concentration in vulnerable tissues. In addition, the liposomes allowmuch smaller doses of drug to be used, thus reducing the side effects of that drug. On the other hand, zinc phthalocyanine (ZnPc) is a potential drug that is being tested as a photosensitizer for photodynamic therapy (PDT). PDT involves the systemic administration of a photosensitizer followed by illumination with light of an appropriate wavelength, which results in the formation of singlet oxygen (O2) for destroying cancer cells. [6] ZnPc has a high absorption coefficient at 650–700 nm with optimal tissue penetration. It has a long lifetime in the triplet excited state, thus resulting in the highly efficient production of O2 which is the main cytotoxic species in PDT. However, it is insoluble in water and must be incorporated into unilamellar liposomes (Figure 1a). The main drawback of liposomes is their instability in biological media, as well as their sensitivity to many external parameters, such as temperature or osmotic pressure. This instability problem affects the application of liposomes in drug/gene delivery and PDT. Attempts have been made to stabilize liposomes by adsorbing some

Self-assembly and mineralization of genetically modifiable biological nanofibers driven by β-structure formation.

Bioinspired mineralization is an innovative approach to the fabrication of bone biomaterials mimicking the natural bone. Bone mineral hydroxylapatite (HAP) is preferentially oriented with c-axis parallel to collagen fibers in natural bone. However, such orientation control is not easy to achieve in artificial bone biomaterials. To overcome the lack of such orientation control, we fabricated a phage-HAP composite by genetically engineering M13 phage, a nontoxic bionanofiber, with two HAP-nucleating peptides derived from one of the noncollagenous proteins, Dentin Matrix Protein-1 (DMP1). The phage is a biological nanofiber that can be mass produced by infecting bacteria and is nontoxic to human beings. The resultant HAP-nucleating phages are able to self-assemble into bundles by forming β-structure between the peptides displayed on their side walls. The β-structure further promotes the oriented nucleation and growth of HAP crystals within the nanofibrous phage bundles with their c-axis preferentially parallel to the bundles. We proposed that the preferred orientation resulted from the stereochemical matching between the negatively charged amino acid residues within the β-structure and the positively charged calcium ions on the (001) plane of HAP crystals. The self-assembly and mineralization driven by the β-structure formation represent a new route for fabricating mineralized fibers that can serve as building blocks in forming bone repair biomaterials and mimic the basic structure of natural bones.

Controlling Nanostructures of Mesoporous Silica Fibers by Supramolecular Assembly of Genetically Modifiable Bacteriophages

A useful virus: The synthesis of a new family of mesoporous silica fibers is reported. Monodisperse filamentous bacteriophages self-assembled into highly ordered hexagonal lattices that were used as templates for the formation of silica nanostructures. Removal of the bacteriophage assembly through calcination led to the formation of mesoporous silica fibers with pore structures precisely defined by the bacteriophage assembly (see picture).

Ultrasensitive rapid detection of human serum antibody biomarkers by biomarker-capturing viral nanofibers.

Candida albicans (C. albicans) infection causes high mortality rates within cancer patients. Due to the low sensitivity of the current diagnosis systems, a new sensitive detection method is needed for its diagnosis. Toward this end, here we exploited the capability of genetically displaying two functional peptides, one responsible for recognizing the biomarker for the infection (antisecreted aspartyl proteinase 2 IgG antibody) in the sera of cancer patients and another for binding magnetic nanoparticles (MNPs), on a single filamentous fd phage, a human-safe bacteria-specific virus. The resultant phage is first decorated with MNPs and then captures the biomarker from the sera. The phage-bound biomarker is then magnetically enriched and biochemically detected. This method greatly increases the sensitivity and specificity of the biomarker detection. The average detection time for each serum sample is only about 6 h, much shorter than the clinically used gold standard method, which takes about 1 week. The detection limit of our nanobiotechnological method is approximately 1.1 pg/mL, about 2 orders of magnitude lower than that of the traditional antigen-based method, opening up a new avenue to virus-based disease diagnosis.

Phage as a Genetically Modifiable Supramacromolecule in Chemistry, Materials and Medicine

Filamentous bacteriophage (phage) is a genetically modifiable supramacromolecule. It can be pictured as a semiflexible nanofiber (∼900 nm long and ∼8 nm wide) made of a DNA core and a protein shell with the former genetically encoding the latter. Although phage bioengineering and phage display techniques were developed before the 1990s, these techniques have not been widely used for chemistry, materials, and biomedical research from the perspective of supramolecular chemistry until recently. Powered by our expertise in displaying a foreign peptide on its surface through engineering phage DNA, we have employed phage to identify target-specific peptides, construct novel organic-inorganic nanohybrids, develop biomaterials for disease treatment, and generate bioanalytical methods for disease diagnosis. Compared with conventional biomimetic chemistry, phage-based supramolecular chemistry represents a new frontier in chemistry, materials science, and medicine. In this Account, we introduce our recent successful efforts in phage-based supramolecular chemistry, by integrating the unique nanofiber-like phage structure and powerful peptide display techniques into the fields of chemistry, materials science, and medicine: (1) successfully synthesized and assembled silica, hydroxyapatite, and gold nanoparticles using phage templates to form novel functional materials; (2) chemically introduced azo units onto the phage to form photoresponsive functional azo-phage nanofibers via a diazotization reaction between aromatic amino groups and the tyrosine residues genetically displayed on phage surfaces; (3) assembled phage into 2D films for studying the effects of both biochemical (the peptide sequences displayed on the phages) and biophysical (the topographies of the phage films) cues on the proliferation and differentiation of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) and identified peptides and topographies that can induce their osteogenic differentiation; (4) discovered that phage could induce angiogenesis and osteogenesis for MSC-based vascularized bone regeneration; (5) identified novel breast cancer cell-targeting and MSC-targeting peptides and used them to significantly improve the efficiency of targeted cancer therapy and MSC-based gene delivery, respectively; (6) employed engineered phage as a probe to achieve ultrasensitive detection of biomarkers from serum of human patients for disease diagnosis; and (7) constructed centimeter-scale 3D multilayered phage assemblies with the potential application as scaffolds for bone regeneration and functional device fabrication. Our findings demonstrated that phage is indeed a very powerful supramacromolecule suitable for not only developing novel nanostructures and biomaterials but also advancing important fields in biomedicine, including molecular targeting, cancer diagnosis and treatment, drug and gene delivery, stem cell fate direction, and tissue regeneration. Our successes in exploiting phage in chemistry, materials, and medicine suggest that phage itself is nontoxic at the cell level and can be safely used for detecting biomarkers in vitro. Moreover, although we have demonstrated successful in vivo tissue regeneration induced by phage, we believe future studies are needed to evaluate the in vivo biodistribution and potential risks of the phage-based biomaterials.

Oxide formation on biological nanostructures via a structure-directing agent: towards an understanding of precise structural transcription

Biomimetic silica formation is strongly dependent on the presence of cationic amine groups which hydrolyze organosilicate precursors and bind to silicate oligomers. Since most biological species possess anionic surfaces, the dependence on amine groups limits utilization of biotemplates for fabricating materials with specific morphologies and pore structures. Here, we report a general aminopropyltriethoxysilane (APTES) directed method for preparing hollow silica with well-defined morphologies using varying biotemplates (proteins, viruses, flagella, bacteria and fungi). Control experiments, pH evolution measurements and 29Si NMR spectroscopic studies have revealed a mechanism of the assembly of APTES on bio-surfaces with subsequent nucleation and growth of silica. The APTES assembly and nuclei formation on bio-surfaces ensured precise transcription of the morphologies of biotemplates to the resulting silica. This method could be extended to the preparation of other oxides.

Identification of microtubule-binding domains on microtubule-associated proteins by major coat phage display technique.

Microtubule is an important structural and functional component in cells. Microtubule-associated proteins (MAPs) are a class of proteins that can bind to microtubules and stabilize them to maintain their functions. However, not all the specific microtubule-binding domains on MAPs are clear. Here we report the study of microtubule-binding domains on MAPs from a new angle by biopanning a new type of phage-displayed random peptide library (called landscape phage library) against purified alpha- and beta-tubulins. In the landscape phage library, billions of fd-tet phage clones are present and a unique 9-mer peptide is fused to each of the approximately 3900 copies of major coat protein (pVIII) in each clone. The affinity-selected peptides derived from the biopanning were analyzed by the receptor ligand contacts (RELIC) suite of programs, which is a bioinformatics tool for combinatorial peptide analysis and identification of protein-ligand interaction sites. By using RELIC, the affinity-selected peptides were shown to have similarity with the sequences of two MAP families (MAP1 and MAP2/tau), thereby identifying putative microtubule-binding domains on these MAPs. The tubulin-binding affinity was also confirmed by using transmission electron microscopy (TEM) to characterize the interaction between affinity-selected tubulin-binding phage and tubulins. Our results confirm some known microtubule-binding domains and identify some new microtubule-binding domains and thus shed light into the mechanism of microtubule-MAPs interactions.