Strain-Promoted Cycloadditions in Lipid Bilayers Triggered by Liposome Fusion.

Due to the variety of roles served by the cell membrane, its composition and structure are complex, making it difficult to study. Bioorthogonal reactions, such as the strain promoted azide-alkyne cycloaddition (SPAAC), are powerful tools for exploring the function of biomolecules in their native environment but have been largely unexplored within the context of lipid bilayers. Here, we developed a new approach to study the SPAAC reaction in liposomal membranes using azide- and strained alkyne-functionalized Förster resonance energy transfer (FRET) dye pairs. This study represents the first characterization of the SPAAC reaction between diffusing molecules inside liposomal membranes. Potential applications of this work include in situ bioorthogonal labeling of membrane proteins, improved understanding of membrane dynamics and fluidity, and the generation of new probes for biosensing assays.

Transforming Nanomaterial Synthesis with Flow Chemistry.

Microfluidic methods for the synthesis of nanomaterials allow the generation of high-quality products with outstanding structural, electronic and optical properties. At a fundamental level, this is engendered by the ability to control both heat and mass transfer in a rapid and precise manner, but also by the facile integration of in-line characterization tools and machine learning algorithms. Such integrated platforms provide for exquisite control over material properties during synthesis, accelerate the optimization of electronic and optical properties and bestow new insights into the optoelectronic properties of nanomaterials. Herein, we present a brief perspective on the role that microfluidic technologies can play in nanomaterial synthesis, with a particular focus on recent studies that incorporate in-line optical characterization and machine learning. We also consider the importance and challenges associated with integrating additional functional components within experimental workflows and the upscaling of microfluidic platforms for production of industrial-scale quantities of nanomaterials.

Microfluidic synthesis of monodisperse and size-tunable CsPbBr3 supraparticles.

The highly controlled, microfluidic template-assisted self-assembly of CsPbBr3 nanocrystals into spherical supraparticles is presented, achieving precise control over average supraparticle size through the variation of nanocrystal concentration and droplet size; thus facilitating the synthesis of highly monodisperse, sub-micron supraparticles (with diameters between 280 and 700 nm).

Noble Metal Nanoparticle Biosensors: From Fundamental Studies toward Point-of-Care Diagnostics.

Noble metal nanoparticles (NMNPs) have become firmly established as effective agents to detect various biomolecules with extremely high sensitivity. This ability stems from the collective oscillation of free electrons and extremely large electric field enhancement under exposure to light, leading to various light-matter interactions such as localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering. A remarkable feature of NMNPs is their customizability by mechanisms such as particle etching, growth, and aggregation/dispersion, yielding distinct color changes and excellent opportunities for colorimetric biosensing in user-friendly assays and devices. They are readily functionalized with a large variety of capping agents and biomolecules, with resultant bioconjugates often possessing excellent biocompatibility, which can be used to quantitatively detect analytes from physiological fluids. Furthermore, they can possess excellent catalytic properties that can achieve significant signal amplification through mechanisms such as the catalytic transformation of colorless substrates to colored reporters. The various excellent attributes of NMNP biosensors have put them in the spotlight for developing high-performance in vitro diagnostic (IVD) devices that are particularly well-suited to mitigate the societal threat that infectious diseases pose. This threat continues to dominate the global health care landscape, claiming millions of lives annually. NMNP IVDs possess the potential to sensitively detect infections even at very early stages with affordable and field-deployable devices, which will be key to strengthening infectious disease management. This has been the major focal point of current research, with a view to new avenues for early multiplexed detection of infectious diseases with portable devices such as smartphones, especially in resource-limited settings.In this Account, we provide an overview of our original inspiration and efforts in NMNP-based assay development, together with some more sophisticated IVD assays by ourselves and many others. Our work in the area has led to our recent efforts in developing IVDs for high-profile infectious diseases, including Ebola and HIV. We emphasize that integration with digital platforms represents an opportunity to establish and efficiently manage widespread testing, tracking, epidemiological intelligence, and data sharing backed by community participation. We highlight how digital technologies can address the limitations of conventional diagnostic technologies at the point of care (POC) and how they may be used to abate and contain the spread of infectious diseases. Finally, we focus on more recent integrations of noble metal nanoparticles with Raman spectroscopy for accurate, noninvasive POC diagnostics with improved sensitivity and specificity.

An amplification-free ultra-sensitive electrochemical CRISPR/Cas biosensor for drug-resistant bacteria detection.

Continued development of high-performance and cost-effective in vitro diagnostic tools is vital for improving infectious disease treatment and transmission control. For nucleic acid diagnostics, moving beyond enzyme-mediated amplification assays will be critical in reducing the time and complexity of diagnostic technologies. Further, an emerging area of threat, in which in vitro diagnostics will play an increasingly important role, is antimicrobial resistance (AMR) in bacterial infections. Herein, we present an amplification-free electrochemical CRISPR/Cas biosensor utilizing silver metallization (termed E-Si-CRISPR) to detect methicillin-resistant Staphylococcus aureus (MRSA). Using a custom-designed guide RNA (gRNA) targeting the mecA gene of MRSA, the Cas12a enzyme allows highly sensitive and specific detection when employed with silver metallization and square wave voltammetry (SWV). Our biosensor exhibits excellent analytical performance, with detection and quantitation limits of 3.5 and 10 fM, respectively, and linearity over five orders of magnitude (from 10 fM to 0.1 nM). Importantly, we observe no degradation in performance when moving from buffer to human serum samples, and achieve excellent selectivity for MRSA in human serum in the presence of other common bacteria. The E-Si-CRISPR method shows significant promise as an ultrasensitive field-deployable device for nucleic acid-based diagnostics, without requiring nucleic acid amplification. Finally, adjustment to a different disease target can be achieved by simple modification of the gRNA protospacer.

Tuning DNA-nanoparticle conjugate properties allows modulation of nuclease activity.

Enzyme-nanoparticle interactions can give rise to a range of new phenomena, most notably significant enzymatic rate enhancement. Accordingly, the careful study and optimization of such systems is likely to give rise to advanced biosensing applications. Herein, we report a systematic study of the interactions between nuclease enzymes and oligonucleotide-coated gold nanoparticles (spherical nucleic acids, SNAs), with the aim of revealing phenomena worthy of evolution into functional nanosystems. Specifically, we study two nucleases, an exonuclease (ExoIII) and an endonuclease (Nt.BspQI), via fluorescence-based kinetic experiments, varying parameters including enzyme and substrate concentrations, and nanoparticle size and surface coverage in non-recycling and a recycling formats. We demonstrate the tuning of nuclease activity by SNA characteristics and show that the modular units of SNAs can be leveraged to either accelerate or suppress nuclease kinetics. Additionally, we observe that the enzymes are capable of cleaving restriction sites buried deep in the oligonucleotide surface layer and that enzymatic rate enhancement occurs in the target recycling format but not in the non-recycling format. Furthermore, we demonstrate a new SNA phenomenon, we term 'target stacking', whereby nucleic acid hybridization efficiency increases as enzyme cleavage proceeds during the beginning of a reaction. This investigation provides important data to guide the design of novel SNAs in biosensing and in vitro diagnostic applications.

Fluorometric Paper-Based, Loop-Mediated Isothermal Amplification Devices for Quantitative Point-of-Care Detection of Methicillin-Resistant Staphylococcus aureus (MRSA).

Loop-mediated isothermal amplification (LAMP) has been widely used to detect many infectious diseases. However, minor inconveniences during the steps of adding reaction ingredients and lack of simple color results hinder point-of-care detection. We therefore invented a fluorometric paper-based LAMP by incorporating LAMP reagents, including a biotinylated primer, onto a cellulose membrane paper, with a simple DNA fluorescent dye incubation that demonstrated rapid and accurate results parallel to quantitative polymerase chain reaction (qPCR) methods. This technology allows for instant paper strip detection of methicillin-resistant Staphylococcus aureus (MRSA) in the laboratory and clinical samples. MRSA represents a major public health problem as it can cause infections in different parts of the human body and yet is resistant to commonly used antibiotics. In this study, we optimized LAMP reaction ingredients and incubation conditions following a central composite design (CCD) that yielded the shortest reaction time with high sensitivity. These CCD components and conditions were used to construct the paper-based LAMP reaction by immobilizing the biotinylated primer and the rest of the LAMP reagents to produce the ready-to-use MRSA diagnostic device. Our paper-based LAMP device could detect as low as 10 ag (equivalent to 1 copy) of the MRSA gene mecA within 36-43 min, was evaluated using both laboratory (individual cultures of MRSA and non-MRSA bacteria) and clinical blood samples to be 100% specific and sensitive compared to qPCR results, and had 35 day stability under 25 °C storage. Furthermore, the color readout allows for quantitation of MRSA copies. Hence, this device is applicable for point-of-care MRSA detection.

In Situ Nucleic Acid Amplification and Ultrasensitive Colorimetric Readout in a Paper-Based Analytical Device Using Silver Nanoplates.

A rapid, highly sensitive, and quantitative colorimetric paper-based analytical device (PAD) based on silver nanoplates (AgNPls) and loop-mediated isothermal amplification (LAMP) is presented. It is shown that cauliflower-like concatemer LAMP products can mediate crystal etching of AgNPls, with a threefold signal enhancement versus linear dsDNA. Methicillin-resistant Staphylococcus aureus (MRSA), an antimicrobial resistant bacterium that poses a formidable risk with persistently high mortality, is used as a model pathogen. Due to the excellent color contrast provided by AgNPls, the PAD allows qualitative analysis by the naked eye and quantitative analysis using a smartphone camera, with detection limits down to a single copy in just 30 min, and a linear response from 1 to 104 copies (R2 = 0.994). The entire assay runs in situ on the paper surface, which drastically simplifies operation of the device. This is the first demonstration of single copy detection using a colorimetric readout, and the developed PAD shows great promise for translation into an ultrasensitive gene-based point-of-care test for any infectious disease target, via modification of the LAMP primer set.

An ultrasensitive non-noble metal colorimetric assay using starch-iodide complexation for Ochratoxin A detection.

Colorimetric sandwich-type biosensors that can both provide sensitivity competitive with fluorescence-based approaches, and leverage reagents that are cost-effective, widely available and as safe as possible, are highly sought after. Herein, we demonstrate an alternative highly-sensitive colorimetric method for paper-based sandwich-type biosensing that uses starch-iodide complexation to simplify practical biosensing using ubiquitous reagents. Targeting the mycotoxin ochratoxin A (OTA), a covalently-immobilised OTA antibody on a cellulose surface captures OTA and forms a sandwich with OTA aptamer-conjugated glucose oxidase. Adding the chromogenic reagents at an optimized concentration, a distinct blue color develops within 30 min, offering excellent contrast with the clear/white of the negative sample. With a sampling volume down to just 5 µL, the assay exhibits concentration limits of detection and quantitation of 20 and 320 pg mL-1, respectively, and a linear range from 10-1 to 105 ng mL-1 (R2 = 0.997). The method displays excellent selectivity against related mycotoxins, excellent %recovery (95-117%) and robust operation in complex matrices (beer, urine and human serum), with no significant difference versus gold-standard liquid chromatography. Along with its excellent analytical performance, this assay benefits from non-toxic and extremely cheap reagents that can be safely disposed of in the field, and presents an attractive alternative to toxic dyes and nanoparticles.

Enzyme-Assisted Nucleic Acid Detection for Infectious Disease Diagnostics: Moving toward the Point-of-Care.

Driven by complex and interconnected factors, including population growth, climate change, and geopolitics, infectious diseases represent one of the greatest healthcare challenges of the 21st century. Diagnostic technologies are the first line of defense in the fight against infectious disease, providing critical information to inform epidemiological models, track diseases, decide treatment choices, and ultimately prevent epidemics. The diagnosis of infectious disease at the genomic level using nucleic acid disease biomarkers has proven to be the most effective approach to date. Such methods rely heavily on enzymes to specifically amplify or detect nucleic acids in complex samples, and significant effort has been exerted to harness the power of enzymes for in vitro nucleic acid diagnostics. Unfortunately, significant challenges limit the potential of enzyme-assisted nucleic acid diagnostics, particularly when translating diagnostic technologies from the lab toward the point-of-use or point-of-care. Herein, we discuss the current state of the field and highlight cross-disciplinary efforts to solve the challenges associated with the successful deployment of this important class of diagnostics at or near the point-of-care.

Long-armed hexapod nanocrystals of cesium lead bromide.

Colloidal lead halide perovskite nanocrystals (LHP NCs) assume a variety of morphologies (e.g. cubes, sheets, and wires). Their labile structural and surface characters allow them to undergo post-synthetic evolution of shape and crystallographic characters. Such transformations can be advantageous or deleterious, and it is therefore vital to both understand and exert control over these processes. In this study, we report novel long-armed hexapod structures of cesium lead bromide nanocrystals. These branched structures evolve from quantum-confined CsPbBr3 nanosheets to Cs4PbBr6 hexapods over a period of 24 hours. Time-resolved optical and structural characterization reveals a post-synthesis mechanism of phase transformation, oriented attachment and branch elongation. More generally, the study reveals important processes associated with LHP NC aging and demonstrates the utility of slow reaction kinetics in obtaining complex morphologies.

Droplet microfluidics: from proof-of-concept to real-world utility?

Droplet microfluidics constitutes a diverse and practical tool set that enables chemical and biological experiments to be performed at high speed and with enhanced efficiency when compared to conventional instrumentation. Indeed, in recent years, droplet-based microfluidic tools have been used to excellent effect in a range of applications, including materials synthesis, single cell analysis, RNA sequencing, small molecule screening, in vitro diagnostics and tissue engineering. Our 2011 Chemical Communications Highlight Article [Chem. Commun., 2011, 47, 1936-1942] reviewed some of the most important technological developments and applications of droplet microfluidics, and identified key challenges that needed to be addressed in the short term. In the current contribution, we consider the intervening eight years, and assess the contributions that droplet-based microfluidics has made to experimental science in its broadest sense.

Rolling Circle Transcription-Amplified Hierarchically Structured Organic-Inorganic Hybrid RNA Flowers for Enzyme Immobilization.

Programmable nucleic acids have emerged as powerful building blocks for the bottom-up fabrication of two- or three-dimensional nano- and microsized constructs. Here we describe the construction of organic-inorganic hybrid RNA flowers (hRNFs) via rolling circle transcription (RCT), an enzyme-catalyzed nucleic acid amplification reaction. These hRNFs are highly adaptive structures with controlled sizes, specific nucleic acid sequences, and a highly porous nature. We demonstrated that hRNFs are applicable as potential biological platforms, where the hRNF scaffold can be engineered for versatile surface functionalization and the inorganic component (magnesium ions) can serve as an enzyme cofactor. For surface functionalization, we proposed robust and straightforward approaches including in situ synthesis of functional hRNFs and postfunctionalization of hRNFs that enable facile conjugation with various biomolecules and nanomaterials (i.e., proteins, enzymes, organic dyes, inorganic nanoparticles) using selective chemistries (i.e., avidin-biotin interaction, copper-free click reaction). In particular, we showed that hRNFs can serve as soft scaffolds for ß-galactosidase immobilization and greatly enhance enzymatic activity and stability. Therefore, the proposed concepts and methodologies are not only fundamentally interesting when designing RNA scaffolds or RNA bionanomaterials assembled with enzymes but also have significant implications on their future utilization in biomedical applications ranging from enzyme cascades to biosensing and drug delivery.

An Exonuclease I-Assisted Silver-Metallized Electrochemical Aptasensor for Ochratoxin A Detection.

Ochratoxin A (OTA)-a mycotoxin produced by Aspergillus and Penicillium fungi-is a carcinogen and common trace contaminant in agricultural and processed food products. As consumption is detrimental to human and animal health, regular product monitoring is vital, and highly sensitive and portable OTA sensors are necessary in many circumstances. Herein, we report an ultrasensitive, electroanalytical aptasensor for precise determination of OTA at trace levels. The sensor leverages a DNA aptamer to capture OTA and silver metallization as a signal enhancer. Exonuclease I is used to digest unbound aptamers, engendering excellent background signal suppression and sensitivity enhancements. Efficient optimization of assay conditions is achieved using central composite design (CCD), allowing rapid evaluation of both the electrode and square wave voltammetry parameter space. The sensor exhibits excellent analytical performance, with a concentration limit of detection of 0.7 pg mL-1, a limit of quantitation of 2.48 pg mL-1, and a linear dynamic range ( R2 = 0.968) of over 6 orders of magnitude (between 1 pg mL-1 and 0.1 µg mL-1). Direct comparison with ultraperformance liquid chromatography (UPLC) indicates excellent analytical performance for standard solutions ( R2 = 0.995) and spiked beer samples ( R2 = 0.993), with almost quantitative recovery and less than 5% relative standard deviation (RSD).

Detection of microRNA biomarkers via inhibition of DNA-mediated liposome fusion.

We report the specific and sensitive detection of microRNA using an inverse DNA-mediated liposome fusion assay. This assay is homogeneous, and does not require washing, separation, or enzyme-associated amplification steps. By fine-tuning the surface functionalisation of the liposomes, liposome concentration, and assay temperature, we demonstrated a sub-nanomolar limit of detection for the target.

A sample-in-digital-answer-out system for rapid detection and quantitation of infectious pathogens in bodily fluids.

A variety of automated sample-in-answer-out systems for in vitro molecular diagnostics have been presented and even commercialized. Although efficient in operation, they are incapable of quantifying targets, since quantitation based on analog analytical methods (via standard curve analysis) is complex, expensive, and challenging. To address this issue, herein, we describe an integrated sample-in-digital-answer-out (SIDAO) diagnostic system incorporating DNA extraction and digital recombinase polymerase amplification, which enables rapid and quantitative nucleic acid analysis from bodily fluids within a disposable cartridge. Inside the cartridge, reagents are pre-stored in sterilized tubes, with an automated pipetting module allowing facile liquid transfer. For digital analysis, we fabricate a simple, single-layer polydimethylsiloxane microfluidic device and develop a novel and simple sample compartmentalization strategy. Sample solution is partitioned into an array of 40,044 fL-volume microwells by sealing the microfluidic device through the application of mechanical pressure. The entire analysis is performed in a portable, fully automated instrument. We evaluate the quantitative capabilities of the system by analyzing Mycobacterium tuberculosis genomic DNA from both spiked saliva and serum samples, and demonstrate excellent analytical accuracy and specificity. This SIDAO system provides a promising diagnostic platform for quantitative nucleic acid testing at the point-of-care. Graphical abstract ᅟ.

Duplex-Specific Nuclease-Amplified Detection of MicroRNA Using Compact Quantum Dot-DNA Conjugates.

Advances in nanotechnology have provided new opportunities for the design of next-generation nucleic acid biosensors and diagnostics. Indeed, combining advances in functional nanoparticles, DNA nanotechnology, and nuclease-enzyme-based amplification can give rise to new assays with advantageous properties. In this work, we developed a microRNA (miRNA) assay using bright fluorescent quantum dots (QDs), simple DNA probes, and the enzyme duplex-specific nuclease. We employed an isothermal target-recycling mechanism, where a single miRNA target triggers the cleavage of many DNA signal probes. The incorporation of DNA-functionalized QDs enabled a quantitative fluorescent readout, mediated by Förster resonance energy transfer (FRET)-based interaction with the DNA signal probes. Our approach splits the reaction in two, performing the enzyme-mediated amplification and QD-based detection steps separately such that each reaction could be optimized for performance of the active components. Target recycling gave ca. 3 orders of magnitude amplification, yielding highly sensitive detection with a limit of 42 fM (or 1.2 amol) of miR-148, with excellent selectivity versus mismatched sequences and other miRNAs. Furthermore, we used an alternative target (miR-21) and FRET pair for direct and absolute quantification of miR-21 in RNA extracts from human cancer and normal cell lines.

Post-polymerisation functionalisation of conjugated polymer backbones and its application in multi-functional emissive nanoparticles.

Backbone functionalisation of conjugated polymers is crucial to their performance in many applications, from electronic displays to nanoparticle biosensors, yet there are limited approaches to introduce functionality. To address this challenge we have developed a method for the direct modification of the aromatic backbone of a conjugated polymer, post-polymerisation. This is achieved via a quantitative nucleophilic aromatic substitution (SNAr) reaction on a range of fluorinated electron-deficient comonomers. The method allows for facile tuning of the physical and optoelectronic properties within a batch of consistent molecular weight and dispersity. It also enables the introduction of multiple different functional groups onto the polymer backbone in a controlled manner. To demonstrate the versatility of this reaction, we designed and synthesised a range of emissive poly(9,9-dioctylfluorene-alt-benzothiadiazole) (F8BT)-based polymers for the creation of mono and multifunctional semiconducting polymer nanoparticles (SPNs) capable of two orthogonal bioconjugation reactions on the same surface.

MicroRNA Detection by DNA-Mediated Liposome Fusion.

Membrane fusion is a process of fundamental importance in biological systems that involves highly selective recognition mechanisms for the trafficking of molecular and ionic cargos. Mimicking natural membrane fusion mechanisms for the purpose of biosensor development holds great potential for amplified detection because relatively few highly discriminating targets lead to fusion and an accompanied engagement of a large payload of signal-generating molecules. In this work, sequence-specific DNA-mediated liposome fusion is used for the highly selective detection of microRNA. The detection of miR-29a, a known flu biomarker, is demonstrated down to 18 nm within 30 min with high specificity by using a standard laboratory microplate reader. Furthermore, one order of magnitude improvement in the limit of detection is demonstrated by using a novel imaging technique combined with an intensity fluctuation analysis, which is coined two-color fluorescence correlation microscopy.