The introduction of immune checkpoint inhibitors has demonstrated significant improvements in survival for subsets of cancer patients. However, they carry significant and sometimes life-threatening ...toxicities. Prompt prediction and monitoring of immune toxicities have the potential to maximise the benefits of immune checkpoint therapy. Herein, we develop a digital nanopillar SERS platform that achieves real-time single cytokine counting and enables dynamic tracking of immune toxicities in cancer patients receiving immune checkpoint inhibitor treatment - broader applications are anticipated in other disease indications. By analysing four prospective cytokine biomarkers that initiate inflammatory responses, the digital nanopillar SERS assay achieves both highly specific and highly sensitive cytokine detection down to attomolar level. Significantly, we report the capability of the assay to longitudinally monitor 10 melanoma patients during immune inhibitor blockade treatment. Here, we show that elevated cytokine concentrations predict for higher risk of developing severe immune toxicities in our pilot cohort of patients.
Exosomes are vesicles which have garnered interest due to their diagnostic and therapeutic potential. Isolation of pure yields of exosomes from complex biological fluids whilst preserving their ...physical characteristics is critical for downstream applications. In this study, we use 100 nm-liposomes from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol as a model system as a model system to assess the effect of exosome isolation protocols on vesicle recovery and size distribution using a single-particle analysis method. We demonstrate that liposome size distribution and ζ-potential are comparable to extracted exosomes, making them an ideal model for comparison studies. Four different purification protocols were evaluated, with liposomes robustly isolated by three of them. Recovered yields varied and liposome size distribution was unaltered during processing, suggesting that these protocols do not induce particle aggregation. This leads us to conclude that the size distribution profile and characteristics of vesicles are stably maintained during processing and purification, suggesting that reports detailing how exosomes derived from tumour cells differ in size to those from normal cells are reporting a real phenomenon. However, we hypothesize that larger particles present in most purified exosome samples represent co-purified contaminating non-exosome debris. These isolation techniques are therefore likely nonspecific and may co-isolate non-exosome material of similar physical properties.
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► The accuracy of particle size distribution measurements is technique dependent. ► The disparity between measurements made by different techniques increases with sample complexity ...and polydispersity. ► Only TRPS and DCS techniques resolved all particles present in complex multimodal samples. ► PTA and DLS techniques had lower resolution and reported higher polydispersity than truly present in all test samples. ► All four techniques tested had high repeat measurement precision.
The particle size distribution (PSD) of a polydisperse or multimodal system can often be difficult to obtain due to the inherent limitations in established measurement techniques. For this reason, the resolution, accuracy and precision of three new and one established, commercially available and fundamentally different particle size analysis platforms were compared by measuring both individual and a mixed sample of monodisperse, sub-micron (220, 330, and 410nm – nominal modal size) polystyrene particles. The platforms compared were the qNano Tunable Resistive Pulse Sensor, Nanosight LM10 Particle Tracking Analysis System, the CPS Instruments’s UHR24000 Disc Centrifuge, and the routinely used Malvern Zetasizer Nano ZS Dynamic Light Scattering system. All measurements were subjected to a peak detection algorithm so that the detected particle populations could be compared to ‘reference’ Transmission Electron Microscope measurements of the individual particle samples. Only the Tunable Resistive Pulse Sensor and Disc Centrifuge platforms provided the resolution required to resolve all three particle populations present in the mixed ‘multimodal’ particle sample. In contrast, the light scattering based Particle Tracking Analysis and Dynamic Light Scattering platforms were only able to detect a single population of particles corresponding to either the largest (410nm) or smallest (220nm) particles in the multimodal sample, respectively. When the particle sets were measured separately (monomodal) each platform was able to resolve and accurately obtain a mean particle size within 10% of the Transmission Electron Microscope reference values. However, the broadness of the PSD measured in the monomodal samples deviated greatly, with coefficients of variation being ∼2–6-fold larger than the TEM measurements across all four platforms. The large variation in the PSDs obtained from these four, fundamentally different platforms, indicates that great care must still be taken in the analysis of samples known to have complex PSDs. All of the platforms were found to have high precision, i.e. they gave rise to less than 5% variance in PSD shape descriptors over the replicate measurements.
Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can ...overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering ∼1 g L
in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories.
Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract ...nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries.
Real-time monitoring of cancer cells' phenotypic evolution during therapy can provide vital tumour biology information for treatment management. Circulating tumour cell (CTC) analysis has emerged as ...a useful monitoring tool, but its routine usage is restricted by either limited multiplexing capability or sensitivity. Here, we demonstrate the use of antibody-conjugated and Raman reporter-coated gold nanoparticles for simultaneous labelling and monitoring of multiple CTC surface markers (named as "cell signature"), without the need for isolating individual CTCs. We observe cell heterogeneity and phenotypic changes of melanoma cell lines during molecular targeted treatment. Furthermore, we follow the CTC signature changes of 10 stage-IV melanoma patients receiving immunological or molecular targeted therapies. Our technique maps the phenotypic evolution of patient CTCs sensitively and rapidly, and shows drug-resistant clones having different CTC signatures of potential clinical value. We believe our proposed method is of general interest in the CTC relevant research and translation fields.
Precision oncology, defined as the use of the molecular understanding of cancer to implement personalized patient treatment, is currently at the heart of revolutionizing oncology practice. Due to the ...need for repeated molecular tumor analyses in facilitating precision oncology, liquid biopsies, which involve the detection of noninvasive cancer biomarkers in circulation, may be a critical key. Yet, existing liquid biopsy analysis technologies are still undergoing an evolution to address the challenges of analyzing trace quantities of circulating tumor biomarkers reliably and cost effectively. Consequently, the recent emergence of cutting‐edge plasmonic nanomaterials represents a paradigm shift in harnessing the unique merits of surface‐enhanced Raman scattering (SERS) biosensing platforms for clinical liquid biopsy applications. Herein, an expansive review on the design/synthesis of a new generation of diverse plasmonic nanomaterials, and an updated evaluation of their demonstrated SERS‐based uses in liquid biopsies, such as circulating tumor cells, tumor‐derived extracellular vesicles, as well as circulating cancer proteins, and tumor nucleic acids is presented. Existing challenges impeding the clinical translation of plasmonic nanomaterials for SERS‐based liquid biopsy applications are also identified, and outlooks and insights into advancing this rapidly growing field for practical patient use are provided.
In a bid to facilitate next‐generation plasmonic nanomaterials for clinical exploration of SERS‐based liquid biopsy applications, a detailed overview of up‐to‐date plasmonic nanomaterials that are designed for SERS applications and newly developed SERS technologies for liquid biopsies, is provided.
The accurate identification and stratified treatment of clinically significant early-stage prostate cancer have been ongoing concerns since the outcomes of large international prostate cancer ...screening trials were reported. The controversy surrounding clinical and cost benefits of prostate cancer screening has highlighted the lack of strategies for discriminating high-risk disease (that requires early treatment) from low-risk disease (that could be managed using watchful waiting or active surveillance). Advances in molecular subtyping and multiomics nanotechnology-based prostate cancer risk delineation can enable refinement of prostate cancer molecular taxonomy into clinically meaningful and treatable subtypes. Furthermore, the presence of intertumoural and intratumoural heterogeneity in prostate cancer warrants the development of novel nanodiagnostic technologies to identify clinically significant prostate cancer in a rapid, cost-effective and accurate manner. Circulating and urinary next-generation prostate cancer biomarkers for disease molecular subtyping and the newest complementary nanodiagnostic platforms for enhanced biomarker detection are promising tools for precision prostate cancer management. However, challenges in merging both aspects and clinical translation still need to be overcome.
Exosomes show promise as noninvasive biomarkers for cancer, but their effective capture and specific detection is a significant challenge. Herein, we report a multiplexed microfluidic device for ...highly specific capture and detection of multiple exosome targets using a tunable alternating current electrohydrodynamic (ac-EHD) methodology, referred to as nanoshearing. In our system, electrical body forces generated by ac-EHD act within nanometers of an electrode surface (i.e., within the electrical layer) to generate nanoscaled fluid flow that enhances the specificity of capture and also reduce nonspecific adsorption of weakly bound molecules from the electrode surface. This approach demonstrates the analysis of exosomes derived from cells expressing human epidermal growth factor receptor 2 (HER2) and prostate specific antigen (PSA), and is also capable of specifically isolating exosomes from breast cancer patient samples. The device also exhibited a 3-fold enhancement in detection sensitivity in comparison to hydrodynamic flow based assays (LOD 2760 exosomes/μL for ac-EHD vs LOD 8300 exosomes/μL for hydrodynamic flow; (n = 3)). We propose this approach can potentially have relevance as a simple and rapid quantification tool to analyze exosome targets in biological applications.
Femtosecond laser ablation is a robust tool for the fabrication of microhole structures. This technique has several advantages compared to other microfabrication strategies for reliably preparing ...microhole structures of high quality and low cost. However, few studies have explored the use of femtosecond laser ablation in plastic materials because of the lack of controllability over the fabrication process in plastics. In particular, the depth profile of microhole structures prepared by conventional laser ablation techniques in plastics cannot be precisely and reproducibly controlled. In this paper, a novel three-dimensional femtosecond laser ablation technique was developed for the rapid fabrication of precise microhole structures in multiple plastics in air. Using a three-step fabrication scheme, microholes demonstrated extremely clean and sharp geometric features. This new technique also enables the precise creation of arbitrary-shaped microwell structures in plastic substrates through a rapid single-step ablation process, without the need for any masks. As a proof of concept for practical applications, precise microhole structures prepared by this novel femtosecond laser ablation technique were exploited for robust resistive-pulse sensing of microparticles.
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