Gold nanostar and graphitic carbon nitride nanocomposite for serotonin detection in biological fluids and human embryonic kidney cell microenvironment.

A nanosensor comprising of gold nanostars (Au-Nstars)-graphitic carbon nitride (g-C3N4) nanocomposite layered on a glassy carbon electrode (GCE) to detect serotonin (ST) in various body fluids has been fabricated. The nanocomposite and the sensing platform have been thoroughly characterized with UV-visible spectroscopy (UV-vis), transmission electron microscopy (TEM), selected area electron diffraction (SAED), energy dispersive X-ray photoelectron spectroscopy (EDX), and electrochemical techniques such as cyclic voltammetry (CV), linear sweep voltammetry (LSV), and electrochemical impedance spectroscopy (EIS). The designed ST detection probe has achieved a linear dynamic range (LDR) in the range 5 × 10-7 and 1 × 10-3 M with a limit of detection (LOD) of 15.1 nM (RSD < 3.3%). The ST detection capability of the fabricated sensor ranges between the normal and several abnormal pathophysiological situations. The sensor effectively detects ST in real matrices such as urine and blood serum, thus, showing its direct diagnostic applicability. Additionally, the sensor has been tested in the microenvironment of human embryonic kidney (HEK) cells to assess the possibility of ST secretion in cell lines. Interferences because of co-existing molecules have been evaluated, and the shelf-life of the fabricated sensor has been obtained as 8 weeks.

pH-Responsive PEGylated Niosomal Nanoparticles as an Active-Targeting Cyclophosphamide Delivery System for Gastric Cancer Therapy.

A PEGylated niosomal formulation of cyclophosphamide (Nio-Cyclo-PEG) was prepared using a central composite design and characterized in terms of drug loading, size distribution, and average size. The stability of formulations was also studied at different conditions. In vitro cytotoxicity of drug delivery formulations was assessed on gastric cancer cells using MTT assay. The mechanism of cytotoxicity was studied at the transcriptional level by real-time PCR on Caspase3, Caspase9, CyclinD, CyclinE, MMP-2, and MMP-9 genes, while apoptosis was investigated with flow cytometry. The anti-metastatic property was evaluated using the scratch method. Propidium iodide staining was used to study the cell cycle. The results indicated that the as-designed nanocarrier exhibited a controlled drug release pattern with improved nanoparticle stability. It was found that the living cancer cells treated with Nio-Cyclo-PEG showed a significant decrease in number when compared with the niosomal carrier without PEG (Nio-Cyclo) and free drug (Cyclo). Moreover, the drug-loaded nanocarrier induced planned death (apoptosis) in the cancer cells through the regulation of Caspase3, Caspase9, CyclinD, CyclinE, MMP-9, and MMP-2 gene expression, indicating that the Nio-Cyclo-PEG formulation could significantly inhibit the cell cycle at the sub G1 phase as well as prevent the migration of cancer cells. In conclusion, Nio-Cyclo-PEG as developed in this study could serve as an active-targeting drug delivery nanocarriers for gastric cancer therapy with high efficacy and minimal side effects on healthy tissues/cells.

Emerging Strategies in Enhancing Singlet Oxygen Generation of Nano-Photosensitizers Toward Advanced Phototherapy.

The great promise of photodynamic therapy (PDT) has thrusted the rapid progress of developing highly effective photosensitizers (PS) in killing cancerous cells and bacteria. To mitigate the intrinsic limitations of the classical molecular photosensitizers, researchers have been looking into designing new generation of nanomaterial-based photosensitizers (nano-photosensitizers) with better photostability and higher singlet oxygen generation (SOG) efficiency, and ways of enhancing the performance of existing photosensitizers. In this paper, we review the recent development of nano-photosensitizers and nanoplasmonic strategies to enhance the SOG efficiency for better PDT performance. Firstly, we explain the mechanism of reactive oxygen species generation by classical photosensitizers, followed by a brief discussion on the commercially available photosensitizers and their limitations in PDT. We then introduce three types of new generation nano-photosensitizers that can effectively produce singlet oxygen molecules under visible light illumination, i.e., aggregation-induced emission nanodots, metal nanoclusters (< 2 nm), and carbon dots. Different design approaches to synthesize these nano-photosensitizers were also discussed. To further enhance the SOG rate of nano-photosensitizers, plasmonic strategies on using different types of metal nanoparticles in both colloidal and planar metal-PS systems are reviewed. The key parameters that determine the metal-enhanced SOG (ME-SOG) efficiency and their underlined enhancement mechanism are discussed. Lastly, we highlight the future prospects of these nanoengineering strategies, and discuss how the future development in nanobiotechnology and theoretical simulation could accelerate the design of new photosensitizers and ME-SOG systems for highly effective image-guided photodynamic therapy.

Gold Nanostars-AIE Theranostic Nanodots with Enhanced Fluorescence and Photosensitization Towards Effective Image-Guided Photodynamic Therapy.

Dual-functional aggregation-induced photosensitizers (AIE-PSs) with singlet oxygen generation (SOG) ability and bright fluorescence in aggregated state have received much attention in image-guided photodynamic therapy (PDT). However, designing an AIE-PS with both high SOG and intense fluorescence via molecular design is still challenging. In this work, we report a new nanohybrid consisting of gold nanostar (AuNS) and AIE-PS dots with enhanced fluorescence and photosensitization for theranostic applications. The spectral overlap between the extinction of AuNS and fluorescence emission of AIE-PS dots (665 nm) is carefully selected using five different AuNSs with distinct localized surface plasmon (LSPR) peaks. Results show that all the AuNSs can enhance the 1O2 production of AIE-PS dots, among which the AuNS with LSPR peak at 585 nm exhibited the highest 1O2 enhancement factor of 15-fold with increased fluorescence brightness. To the best of our knowledge, this is the highest enhancement factor reported for the metal-enhanced singlet oxygen generation systems. The Au585@AIE-PS nanodots were applied for simultaneous fluorescence imaging and photodynamic ablation of HeLa cancer cells with strongly enhanced PDT efficiency in vitro. This study provides a better understanding of the metal-enhanced AIE-PS nanohybrid systems, opening up new avenue towards advanced image-guided PDT with greatly improved efficacy.

Insights and Perspectives Regarding Nanostructured Fluorescent Materials toward Tackling COVID-19 and Future Pandemics.

The COVID-19 outbreak has exposed the world's preparation to fight against unknown/unexplored infectious and life-threatening pathogens. The unavailability of vaccines, slow or sometimes unreliable real-time virus/bacteria detection techniques, insufficient personal protective equipment (PPE), and a shortage of ventilators and many other transportation equipments have further raised serious concerns. Material research has been playing a pivotal role in developing antimicrobial agents for water treatment and photodynamic therapy, fast and ultrasensitive biosensors for virus/biomarkers detection, as well as for relevant biomedical and environmental applications. It has been noticed that these research efforts nowadays primarily focus on the nanomaterials-based platforms owing to their simplicity, reliability, and feasibility. In particular, nanostructured fluorescent materials have shown key potential due to their fascinating optical and unique properties at the nanoscale to combat against a COVID-19 kind of pandemic. Keeping these points in mind, this review attempts to give a perspective on the four key fluorescent materials of different families, including carbon dots, metal nanoclusters, aggregation-induced-emission luminogens, and MXenes, which possess great potential for the development of ultrasensitive biosensors and infective antimicrobial agents to fight against various infections/diseases. Particular emphasis has been given to the biomedical and environmental applications that are linked directly or indirectly to the efforts in combating COVID-19 pandemics. This review also aims to raise the awareness of researchers and scientists across the world to utilize such powerful materials in tackling similar pandemics in future.

Metal Nanoparticles-Enhanced Biosensors: Synthesis, Design and Applications in Fluorescence Enhancement and Surface-enhanced Raman Scattering.

Metal nanoparticles (NP) that exhibit localized surface plasmon resonance play an important role in metal-enhanced fluorescence (MEF) and surface-enhanced Raman scattering (SERS). Among the optical biosensors, MEF and SERS stand out to be the most sensitive techniques to detect a wide range of analytes from ions, biomolecules to macromolecules and microorganisms. Particularly, anisotropic metal NPs with strongly enhanced electric field at their sharp corners/edges under a wide range of excitation wavelengths are highly suitable for developing the ultrasensitive plasmon-enhanced biosensors. In this review, we first highlight the reliable methods for the synthesis of anisotropic gold NPs and silver NPs in high yield, as well as their alloys and composites with good control of size and shape. It is followed by the discussion of different sensing mechanisms and recent advances in the MEF and SERS biosensor designs. This includes the review of surface functionalization, bioconjugation and (directed/self) assembly methods as well as the selection/screening of specific biorecognition elements such as aptamers or antibodies for the highly selective bio-detection. The right combinations of metal nanoparticles, biorecognition element and assay design will lead to the successful development of MEF and SERS biosensors targeting different analytes both in-vitro and in-vivo. Finally, the prospects and challenges of metal-enhanced biosensors for future nanomedicine in achieving ultrasensitive and fast medical diagnostics, high-throughput drug discovery as well as effective and reliable theranostic treatment are discussed.

Protein-protected gold/silver alloy nanoclusters in metal-enhanced singlet oxygen generation and their correlation with photoluminescence.

Photoluminescent noble metal nanoclusters (NCs, core size <2 nm) have recently emerged as a new type of photosensitizers advantageous over conventional photosensitizers due to their high singlet oxygen (1O2) generation efficiency, excellent photostability and water solubility, as well as good biocompatibility for photodynamic therapy and bioimaging. However, no correlation has been established between the intrinsic 1O2 generation and photoluminescence properties of metal NCs with their size, composition, and concentration, which is important to customize the molecule-like properties of NCs for different applications. Herein, we report a systematic study to uncover the rational design of bimetallic NCs with controllable 1O2 generation efficiency by tuning their compositions through spontaneous galvanic displacement reaction. A series of ultrasmall gold/silver alloy nanoclusters (AuAgNCs) were synthesized by reacting bovine serum albumin (BSA) protein-protected Ag13NCs (13 Ag atoms/cluster) with varying concentrations of gold precursor at room temperature. It was found that the 1O2 generation efficiency of the resultant BSA-protected AuAgNCs were inversely correlated to their photoluminescence intensity. Interestingly, plasmonic gold nanoparticles (>10 nm) were also formed simultaneously by photobleaching of the BSA-AuAgNCs, leading to significant metal enhancement effect to the 1O2 generation rate much higher (~45 times) than that of the monometallic BSA-Ag13NC. This versatile two-for-one strategy to develop next generation metal-enhanced bimetallic NC photosensitizers in one pot opens up new opportunities in designing advanced hybrid nanomaterials with complementary and/or enhanced functionalities.

Simulation guided design of silver nanostructures for plasmon-enhanced fluorescence, singlet oxygen generation and SERS applications.

Plasmonic nanostructures such as gold and silver could alter the intrinsic properties of fluorophores, photosensitizers or Raman reporters in their close vicinity. In this study, we have conducted systematic simulations to provide insight for the design of silver nanostructures with appropriate geometrical features for metal-enhanced fluorescence (MEF), metal-enhanced singlet oxygen generation (ME-SOG) and surface-enhanced Raman scattering (SERS) applications. The size-dependent optical properties and electric field enhancement of single and dimeric nanocubes were simulated. The extinction spectra of silver nanocubes were analysed by the multipole expansion method. Results show that a suitable size of Ag nanocubes for MEF and ME-SOG can be selected based on their maximum light scattering yield, the excitation and emission wavelengths of a particular fluorophore/photosensitizer and their maximum spectral overlap. Simulations of the 'hot-spot' or gap distance between two silver nanocubes with different configurations (i.e., face-to-face, edge-to-edge and corner-to-corner) were also performed. A direct correlation was found between the size and enhanced electric field around the Ag nanocubes simulated under 15 common Raman laser wavelengths from the UV to near-infrared region. The maximum SERS enhancement factor can be achieved by selecting the silver nanocubes with the right orientation, suitable edge length and gap distance that give the highest electric field at a specific Raman laser wavelength. It was also found that the higher order of silver nanostructures, e.g., trimer and tetramer, can lead to better enhancement effects. These simulation results can serve as generic guidelines to rationally design metal-enhancement systems including MEF, ME-SOG and SERS for different application needs without cumbersome optimization and tedious trial-and-error experimentation.

Noncovalent Fluorescent Biodot-Protein Conjugates with Well-Preserved Native Functions for Improved Sweat Glucose Detection.

To overcome the traditional issues of protein labeling, we report herein an effective approach for noncovalent conjugation of the biomolecule-derived fluorescent nanodots (biodot) to functional proteins without the addition of chemical linkers for biosensor development. The as-prepared fluorescent biodot-protein conjugates are very stable near physiological pH, exhibiting excellent photostability and thermal stability. More importantly, the native functions of proteins, including drug binding and enzymatic activities, are well-preserved after conjugating with biodots. The optimized protein conjugation strategy is then applied to prepare biodot-glucose oxidase (GOx) fluorescent sensing probes for sweat glucose detection. Results show that the as-prepared sensing probes could achieve better assay performance than those covalent conjugates as demonstrated herein. Specifically, GOx in the noncovalently bound conjugates are able to catalyze the oxidation of glucose effectively, which generates hydrogen peroxide as a byproduct. In the presence of Fe2+, Fenton reaction takes place to produce hydroxyl radicals and Fe3+, leading to significant fluorescence quenching of biodots on the conjugates. This simple one-step enzymatic assay in a single probe achieves a wide linear range of 25-1000 µM (R2 = 0.99) with a low detection limit of 25 µM. Furthermore, negligible interference is observed in the complex artificial sweat sample for accurate glucose quantification, achieving an excellent recovery rate of 100.5 ± 2.2%. This work provides a facile conjugation method that is generally applicable to a wide range of proteins, which will help to accelerate future development of multifunctional fluorescent probes to provide optical signals with unique protein functions (e.g., enzymatic, recognition, etc.) for biomedical sensing and imaging.

Metal-enhancement study of dual functional photosensitizers with aggregation-induced emission and singlet oxygen generation.

Photosensitizers with aggregation-induced emission (AIE-PS) are attractive for image-guided photodynamic therapy due to their dual functional role in generating singlet oxygen and producing high fluorescent signal in the aggregated state. However, their brightness and treatment efficiency maybe limited in current practice. Herein we report the first systematic investigation on the metal-enhanced fluorescence (MEF) and singlet oxygen generation (ME-SOG) ability of our newly synthesized AIE-photosensitizers. The Ag@AIE-PS of varied sizes were prepared via layer-by-layer assembly with controlled distance between silver nanoparticles (AgNPs) and AIE-PS. A maximum of 6-fold enhancement in fluorescence and 2-fold increment in SOG were observed for the 85nmAg@AIE-PS. Comprehensive characterization and simulation were conducted to unravel the plasmon-enhancement mechanisms of Ag@AIE-PS. Results show that MEF of AIE-PS is determined by the enhanced electric field around AgNPs, while ME-SOG is dictated by the scattering efficiency of the metal core, where bigger AgNPs would result in larger enhancement factor. Furthermore, the optimum distance between AgNPs and AIE-PS to achieve maximum SOG enhancement is shorter than that required for the highest MEF. The correlation of MEF and ME-SOG found in this study is useful for designing new a generation of AIE-photosensitizers with high brightness and treatment efficiency towards practical theranostic application in the future.

Establishing empirical design rules of nucleic acid templates for the synthesis of silver nanoclusters with tunable photoluminescence and functionalities towards targeted bioimaging applications.

DNA-templated silver nanoclusters (AgNCs) are an emerging class of ultrasmall (<2 nm) fluorophores with increasing popularity for bioimaging due to their facile synthesis and tunable emission color. However, design rules correlating different nucleotide sequences with the photoemission properties of AgNCs are still largely unknown, preventing the rational design of DNA templates to fine-tune the emission color, brightness and functionalities of AgNCs for any targeted applications. Herein, we report a systematic investigation to understand the empirical influences of the four basic DNA nucleotides on AgNC synthesis and their effects on photoluminescence properties. After establishing the importance of nucleotide-Ag+ binding and AgNC encapsulation within DNA tetraplex structures, we then determined the unique attributes of each individual nucleobase using different combinations of systematically varied DNA templates. Using the empirical design rules established herein, we were able to predict the photoluminescence behaviours of AgNCs templated by complex aptamer sequences with specific binding affinity to human cancer cells, and to deliberately control their emission color by rational modifications of the DNA template sequences for targeted bioimaging. Our empirical findings from this systematic experimentation can contribute towards the rational design of DNA sequences to customise the photoluminescence properties and biofunctionalities of DNA-protected AgNCs towards multicolour targeted bioimaging applications.

Recent development of nucleic acid nanosensors to detect sequence-specific binding interactions: From metal ions, small molecules to proteins and pathogens.

DNA carries important genetic instructions and plays vital roles in regulating biological activities in living cells. Proteins such as transcription factors binds to DNA to regulate the biological functions of DNA, and similarly many drug molecules also bind to DNA to modulate its functions. Due to the importance of protein-DNA and drug-DNA binding, there has been intense effort in developing novel nanosensors in the same length scale as DNA, to effectively study these binding interactions in details. In addition, aptamers can be artificially selected to detect metal ions and pathogens such as bacteria and viruses, making nucleic acid nanosensors more versatile in detecting a large variety of analytes. In this minireview, we first explained the different types and binding modes of protein-DNA and drug-DNA interactions in the biological systems, as well as aptamer-target binding. This was followed by the review of five types of nucleic acid nanosensors based on optical or electrochemical detection. The five types of nucleic acid nanosensors utilizing colorimetric, dynamic light scattering (DLS), surface-enhanced Raman spectroscopy (SERS), fluorescence and electrochemical detections have been recently developed to tackle some of the challenges in high-throughput screening technology for large scale analysis, which is especially useful for drug development and mass screening for pandemic outbreak such as SARS or COVID-19.

Colorimetric biosensors for point-of-care virus detections.

Colorimetric biosensors can be used to detect a particular analyte through color changes easily by naked eyes or simple portable optical detectors for quantitative measurement. Thus, it is highly attractive for point-of-care detections of harmful viruses to prevent potential pandemic outbreak, as antiviral medication must be administered in a timely fashion. This review paper summaries existing and emerging techniques that can be employed to detect viruses through colorimetric assay design with detailed discussion of their sensing principles, performances as well as pros and cons, with an aim to provide guideline on the selection of suitable colorimetric biosensors for detecting different species of viruses. Among the colorimetric methods for virus detections, loop-mediated isothermal amplification (LAMP) method is more favourable for its faster detection, high efficiency, cheaper cost, and more reliable with high reproducible assay results. Nanoparticle-based colorimetric biosensors, on the other hand, are most suitable to be fabricated into lateral flow or lab-on-a-chip devices, and can be coupled with LAMP or portable PCR systems for highly sensitive on-site detection of viruses, which is very critical for early diagnosis of virus infections and to prevent outbreak in a swift and controlled manner.

Development of Blood-Cell-Selective Fluorescent Biodots for Lysis-Free Leukocyte Imaging and Differential Counting in Whole Blood.

Complete blood count with leukocyte (white blood cell, WBC) differential is one of the most frequently ordered clinical test for disease diagnosis. Herein, multifunctional fluorescent carbon dots derived from biomolecules (biodots) for rapid lysis-free whole blood analysis are developed. Specifically, two types of biodots are molecularly engineered through hydrothermal synthesis for differential blood cells labeling. Type I biodots synthesized from amino acid (serine/threonine) precursors and passivated with polyethylenimine can label both red blood cells (RBCs) and WBCs with excellent contrast in fluorescence intensity, enabling direct counting of leukocytes in whole blood samples without a tedious RBC lysis step. It also allows three-part leukocyte differential counting by flow cytometry without using expensive fluorophore-conjugated antibodies. On the other hand, Type II biodots synthesized from the same amino acid precursors but passivated with a biopolymer (chitosan) are able to selectively lyse RBCs with greater than 98% efficiency to allow simultaneous fluorescent labeling of leukocytes for WBC counting in whole blood. It is envisioned that these novel nanoreagents, which eliminate the cumbersome lysis and antibody conjugation steps for selective labeling of different blood cells, would revolutionize disease diagnostics toward achieving faster, cheaper, and more accurate whole blood analyses in one test.

Nanodot-Directed Formation of Plasmonic-Fluorescent Nanohybrids toward Dual Optical Detection of Glucose and Cholesterol via Hydrogen Peroxide Sensing.

Hybrid nanoparticles (NPs) have emerged as an important class of nanomaterials owing to their integrated enhanced properties and functionality. In this study, we have developed an effective nanodot templating strategy for the in situ formation of surfactant-free nanohybrids with unique plasmonic-fluorescent properties. A bright photoluminescent biodot synthesized from serine and histamine biomolecular precursors (Ser-Hist dot) was first engineered to have rich functional groups on the nanosurface capable of anchoring Ag+ ions via electrostatic interaction. Upon UV irradiation, free electrons could transfer from the photoexcited Ser-Hist dot to the Ag+ ions, facilitating the in situ growth of AgNPs. The resulting nanohybrid system (Bio@AgNPs) exhibits distinct characteristic surface plasmon resonance absorbance and highly quenched PL intensity due to the inner filter effect. Furthermore, the Bio@AgNP nanohybrid retains its redox capability, enabling hydrogen peroxide sensing via AgNP etching, which in turn empowers a dual colorimetric and fluorescent detection of glucose and cholesterol in complex biological samples (i.e., synthetic urine and human plasma) with high selectivity and sensitivity. This finding reveals a new effective and facile method for the preparation of highly functional hybrid nanomaterials for dual-mode detection of hydrogen peroxide-producing species and/or reactions.

Seeds, fermented foods, and agricultural by-products as sources of plant-derived antibacterial peptides.

The emergence of bacterial resistance against conventional antibiotics and the growing interest in developing alternative, natural antibacterial agents have prompted the search for plant-derived antibacterial peptides in recent decades. Different classes of endogenous antibacterial peptides have been identified from various plant species. Moreover, protein hydrolysates and hydrolysate-derived peptides with potent antibacterial effects have also been identified from numerous plant sources. Antibacterial peptides are often cationic and amphipathic, consisting of fewer than 100 amino acids. They are able to disrupt bacterial membrane integrity via pore formation and/or compromise bacterial metabolic processes. In this review, we summarize current knowledge on the characteristics and modes of action of antibacterial peptides, as well as salient points concerning the production of antibacterial protein hydrolysates from plant proteins. Examples of plant-derived antibacterial hydrolysates and peptides will be highlighted, with particular attention to less explored seeds, fermented plant foods and agricultural by-products. Promising future research directions with regards to the application of plant-derived antibacterial hydrolysates and peptides in food preservation, farm animal disease management, and nutraceutical/functional food development will be proposed.

Nucleotide-derived theranostic nanodots with intrinsic fluorescence and singlet oxygen generation for bioimaging and photodynamic therapy.

Nucleic acids are important molecules of life and have recently emerged as important functional materials to synthesize, organize and assemble inorganic nanoparticles for various technological applications. In this study, we have systematically investigated the four basic nucleotides of DNA as precursors to form fluorescent nucleotide derived biodots (N-dots) with unique singlet oxygen generation properties by one-pot hydrothermal synthesis. It has been discovered for the first time that the nitrogenous base adenine accounts for the bright fluorescence, while the sugar and phosphate groups of the nucleotide endow the N-dots with good photo-stability. Among the N-dots synthesized in this study, adenosine triphosphate (ATP)-dots were found to exhibit the highest fluorescence quantum yield (QY) of 13.9%, whereas adenosine diphosphate (ADP)-dots exhibited the best photo-stability maintaining 97.6% photoluminescence intensity after continuous UV excitation for 30 min. Overall, deoxyadenosine monophosphate (dAMP)-dots display both high fluorescence QY (12.4%) and good photo-stability (91.9%). Most critically, dAMP-dots show the highest singlet oxygen generation with a remarkable singlet oxygen (1O2) quantum yield of 1.20 surpassing the 1O2 quantum yield of the conventional photosensitizer Rose Bengal (0.75). Further cellular experiments reveal that dAMP-dots possess excellent cellular uptake ability for successful fluorescent labeling with the ability to kill >60% HeLa cancer cells under white light treatment within 10 minutes. Additionally, N-dots possess excellent stability against both UV irradiation and DNase enzymatic action. These results demonstrate the unique physiochemical properties of N-dots, including an ultra-small size for cellular uptake, tunable photoluminescence for bioimaging, excellent aqueous solubility, high chemical stability and photo-stability as well as excellent singlet oxygen quantum yield with inherent biocompatibility for photodynamic therapy, which are important factors contributing to the promising theranostic applications in future personalized nanomedicine.

Rapid colorimetric detection of p53 protein function using DNA-gold nanoconjugates with applications for drug discovery and cancer diagnostics.

The tumor suppressor protein p53 plays a central role in preventing cancer through interaction with DNA response elements (REs) to regulate target gene expression in cells. Due to its significance in cancer biology, relentless efforts have been directed toward understanding p53-DNA interactions for the development of cancer therapeutics and diagnostics. In this paper, we report a rapid, label-free and versatile colorimetric assay to detect wildtype p53 DNA-binding function in complex solutions. The assay design is based on a concept that alters interparticle-distances between RE-AuNPs from a crosslinking effect induced through tetramerization of wildtype p53 protein (p53-WT) upon binding to canonical DNA motifs modified on gold nanoparticles (RE-AuNPs). This leads to a visible solution color change from red to blue, which is quantifiable by the UV- visible absorption spectra with a detection limit of 5 nM. Contrastingly, no color change was observed for the binding-deficient p53 mutants and non-specific proteins due to their inability to crosslink RE-AuNPs. Based on this sensing principle, we further demonstrate its utility for fast detection of drug-induced DNA binding function to cancer-associated Y220C mutant p53 protein using well-established reactivating compounds. By exploiting the dominant-negative property of mutant p53 over p53-WT and interactions with RE-AuNPs, this assay is configurable to detect low numbers of mutant p53 expressing cells in miniscule sample fractions obtained from typical core needle biopsy-sized tissues without signal attrition, alluding to the potential for biopsy sampling in cancer diagnostics or for defining cancer margins. This nanogold enabled colorimetric assay provides a facile yet robust method for studying important parameters influencing p53-DNA interactions with great promises for clinically pertinent applications.

Uncovering the Design Principle of Amino Acid-Derived Photoluminescent Biodots with Tailor-Made Structure-Properties and Applications for Cellular Bioimaging.

Natural amino acids possess side chains with different functional groups (R groups), which make them excellent precursors for programmable synthesis of biomolecule-derived nanodots (biodots) with desired properties. Herein, we report the first systematic study to uncover the material design rules of biodot synthesis from 20 natural α-amino acids via a green hydrothermal approach. The as-synthesized amino acid biodots (AA dots) are comprehensively characterized to establish a structure-property relationship between the amino acid precursors and the corresponding photoluminescent properties of AA dots. It was found that the amino acids with reactive R groups, including amine, hydroxyl, and carboxyl functional groups form unique C-O-C/C-OH and N-H bonds in the AA dots which stabilize the surface defects, giving rise to brightly luminescent AA dots. Furthermore, the AA dots were found to be amorphous and the length of the R group was observed to affect the final morphology (e.g., disclike nanostructure, nanowire, or nanomesh) of the AA dots, which in turn influence their photoluminescent properties. It is noteworthy to highlight that the hydroxyl-containing amino acids, that is, Ser and Thr, form the brightest AA dots with a quantum yield of 30.44% and 23.07%, respectively, and possess high photostability with negligible photobleaching upon continuous UV exposure for 3 h. Intriguingly, by selective mixing of Ser or Thr with another amino acid precursor, the resulting mixed AA dots could inherit unique properties such as improved photostability and significant red shift in their emission wavelength, producing enhanced green and red fluorescent intensity. Moreover, our cellular studies demonstrate that the as-synthesized AA dots display outstanding biocompatibility and excellent intracellular uptake, which are highly desirable for imaging applications. We envision that the material design rules discovered in this study will be broadly applicable for the rational selection of amino acid precursors in the tailored synthesis of biodots.

Locked Nucleic Acid Gapmers and Conjugates Potently Silence ADAM33, an Asthma-Associated Metalloprotease with Nuclear-Localized mRNA.

Two mechanisms dominate the clinical pipeline for oligonucleotide-based gene silencing, namely, the antisense approach that recruits RNase H to cleave target RNA and the RNAi approach that recruits the RISC complex to cleave target RNA. Multiple chemical designs can be used to elicit each pathway. We compare the silencing of the asthma susceptibility gene ADAM33 in MRC-5 lung fibroblasts using four classes of gene silencing agents, two that use each mechanism: traditional duplex small interfering RNAs (siRNAs), single-stranded small interfering RNAs (ss-siRNAs), locked nucleic acid (LNA) gapmer antisense oligonucleotides (ASOs), and novel hexadecyloxypropyl conjugates of the ASOs. Of these designs, the gapmer ASOs emerged as lead compounds for silencing ADAM33 expression: several gapmer ASOs showed subnanomolar potency when transfected with cationic lipid and low micromolar potency with no toxicity when delivered gymnotically. The preferential susceptibility of ADAM33 mRNA to silencing by RNase H may be related to the high degree of nuclear retention observed for this mRNA. Dynamic light scattering data showed that the hexadecyloxypropyl ASO conjugates self-assemble into clusters. These conjugates showed reduced potency relative to unconjugated ASOs unless the lipophilic tail was conjugated to the ASO using a biocleavable linkage. Finally, based on the lead ASOs from (human) MRC-5 cells, we developed a series of homologous ASOs targeting mouse Adam33 with excellent activity. Our work confirms that ASO-based gene silencing of ADAM33 is a useful tool for asthma research and therapy.