Isolation of acute myeloid leukemia blasts from blood using a microfluidic device.

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and associated with poor prognosis. Unfortunately, most of the patients that achieve clinical complete remission after the treatment will ultimately relapse due to the persistence of minimal residual disease (MRD), that is not measurable using conventional technologies in the clinic. Microfluidics is a potential tool to improve the diagnosis by providing early detection of MRD. Herein, different designs of microfluidic devices were developed to promote lateral and vertical mixing of cells in microchannels to increase the contact area of the cells of interest with the inner surface of the device. Possible interactions between the cells and the surface were studied using fluid simulations. For the isolation of leukemic blasts, a positive selection strategy was used, targeting the cells of interest using a panel of specific biomarkers expressed in immature and aberrant blasts. Finally, once the optimisation was complete, the best conditions were used to process patient samples for downstream analysis and benchmarking, including phenotypic and genetic characterisation. The potential of these microfluidic devices to isolate and detect AML blasts may be exploited for the monitoring of AML patients at different stages of the disease.

Advances in Microfluidics for the Implementation of Liquid Biopsy in Clinical Routine.

In recent years, we have seen major advances in the field of liquid biopsy and its implementation in the clinic, mainly driven by breakthrough developments in the area of molecular biology. New developments have seen an integration of microfluidics and also biosensors in liquid biopsy systems, bringing advantages in terms of cost, sensitivity and automation. Without a doubt, the next decade will bring the clinical validation and approval of these combined solutions, which is expected to be crucial for the wide implementation of liquid biopsy systems in clinical routine.

Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.

In Vitro Evaluation of Lipopolyplexes for Gene Transfection: Comparing 2D, 3D and Microdroplet-Enabled Cell Culture.

Complexes combining nucleic acids with lipids and polymers (lipopolyplexes) show great promise for gene therapy since they enable compositional, physical and functional versatility to be optimized for therapeutic efficiency. When developing lipopolyplexes for gene delivery, one of the first evaluations performed is an in vitro transfection efficiency experiment. Many different in vitro models can be used, and the effect of the model on the experiment outcome has not been thoroughly studied. The objective of this work was to compare the insights obtained from three different in vitro models, as well as the potential limitations associated with each of them. We have prepared a series of lipopolyplex formulations with three different cationic polymers (poly-l-lysine, bioreducible poly-l-lysine and polyethyleneimine), and assessed their in vitro biological performance in 2D monolayer cell culture, 3D spheroid culture and microdroplet-based single-cell culture. Lipopolyplexes from different polymers presented varying degrees of transfection efficiency in all models. The best-performing formulation in 2D culture was the polyethyleneimine lipopolyplex, while lipoplexes prepared with bioreducible poly-l-lysine were the only ones achieving any transfection in microdroplet-enabled cell culture. None of the prepared formulations achieved significant gene transfection in 3D culture. All of the prepared formulations were well tolerated by cells in 2D culture, while at least one formulation (poly-l-lysine polyplex) delayed 3D spheroid growth. These results highlight the need for selecting the appropriate in vitro model depending on the intended application.

Label-Free Analysis and Sorting of Microalgae and Cyanobacteria in Microdroplets by Intrinsic Chlorophyll Fluorescence for the Identification of Fast Growing Strains.

Microalgae and cyanobacteria are promising organisms for sustainable biofuel production, but several challenges remain to make this economically viable, including identification of optimized strains with high biomass productivity. Here we report on a novel methodology for the label-free screening and sorting of cyanobacteria and microalgae in a microdroplet platform. We show for the first time that chlorophyll fluorescence can be used to measure differences in biomass between populations of picoliter microdroplets containing different species of cyanobacteria, Synechocystis PCC 6803 and Synechococcus PCC 7002, which exhibit different growth dynamics in bulk culture. The potential and robustness of this label-free screening approach is further demonstrated by the screening and sorting of cells of the green alga Chlamydomonas reinhardtii encapsulated in droplets.

Quality of Life of Colorectal Cancer Survivors: Mapping the Key Indicators by Expert Consensus and Measures for Their Assessment.

Quality of life (QoL) assessments are integral to cancer care, yet their effectiveness in providing essential information for supporting survivors varies. This study aimed to elucidate key indicators of QoL among colorectal cancer survivors from the perspective of healthcare professionals, and to evaluate existing QoL questionnaires in relation to these indicators. Two studies were conducted: a Delphi study to identify key QoL indicators and a scoping review of questionnaires suitable for colorectal cancer survivors. Fifty-four healthcare professionals participated in the Delphi study's first round, with 25 in the second. The study identified two primary QoL domains (physical and psychological) and 17 subdomains deemed most critical. Additionally, a review of 12 questionnaires revealed two instruments assessing the most important general domains. The findings underscored a misalignment between existing assessment tools and healthcare professionals' clinical priorities in working with colorectal cancer survivors. To enhance support for survivors' QoL, efforts are needed to develop instruments that better align with the demands of routine QoL assessment in clinical practice.

Current and Emerging Techniques for Diagnosis and MRD Detection in AML: A Comprehensive Narrative Review.

Acute myeloid leukemia (AML) comprises a group of hematologic neoplasms characterized by abnormal differentiation and proliferation of myeloid progenitor cells. AML is associated with poor outcome due to the lack of efficient therapies and early diagnostic tools. The current gold standard diagnostic tools are based on bone marrow biopsy. These biopsies, apart from being very invasive, painful, and costly, have low sensitivity. Despite the progress uncovering the molecular pathogenesis of AML, the development of novel detection strategies is still poorly explored. This is particularly important for patients that check the criteria for complete remission after treatment, since they can relapse through the persistence of some leukemic stem cells. This condition, recently named as measurable residual disease (MRD), has severe consequences for disease progression. Hence, an early and accurate diagnosis of MRD would allow an appropriate therapy to be tailored, improving a patient's prognosis. Many novel techniques with high potential in disease prevention and early detection are being explored. Among them, microfluidics has flourished in recent years due to its ability at processing complex samples as well as its demonstrated capacity to isolate rare cells from biological fluids. In parallel, surface-enhanced Raman scattering (SERS) spectroscopy has shown outstanding sensitivity and capability for multiplex quantitative detection of disease biomarkers. Together, these technologies can allow early and cost-effective disease detection as well as contribute to monitoring the efficiency of treatments. In this review, we aim to provide a comprehensive overview of AML disease, the conventional techniques currently used for its diagnosis, classification (recently updated in September 2022), and treatment selection, and we also aim to present how novel technologies can be applied to improve the detection and monitoring of MRD.

Label-Free Sensing with Metal Nanostructure-Based Surface-Enhanced Raman Spectroscopy for Cancer Diagnosis.

Surface-Enhanced Raman Spectroscopy (SERS) is a powerful analytical technique for the detection of small analytes with great potential for medical diagnostic applications. Its high sensitivity and excellent molecular specificity, which stems from the unique fingerprint of molecular species, have been applied toward the detection of different types of cancer. The noninvasive and rapid detection offered by SERS highlights its applicability for point-of-care (PoC) deployment for cancer diagnosis, screening, and staging, as well as for predicting tumor recurrence and treatment monitoring. This review provides an overview of the progress in label-free (direct) SERS-based chemical detection for cancer diagnosis with the main focus on the advances in the design and preparation of SERS substrates on the basis of metal nanoparticle structures formed via bottom-up strategies. It begins by introducing a synopsis of the working principles of SERS, including key chemometric approaches for spectroscopic data analysis. Then it introduces the advances of label-free sensing with SERS in cancer diagnosis using biofluids (blood, urine, saliva, sweat) and breath as the detection media. In the end, an outlook of the advances and challenges in cancer diagnosis via SERS is provided.

Discriminating Epithelial to Mesenchymal Transition Phenotypes in Circulating Tumor Cells Isolated from Advanced Gastrointestinal Cancer Patients.

Gastrointestinal (GI) cancers constitute a group of highest morbidity worldwide, with colorectal cancer (CRC) and gastric cancer being among the most frequently diagnosed. The majority of gastrointestinal cancer patients already present metastasis by the time of diagnosis, which is widely associated with cancer-related death. Accumulating evidence suggests that epithelial-to-mesenchymal transition (EMT) in cancer promotes circulating tumor cell (CTCs) formation, which ultimately drives metastasis development. These cells have emerged as a fundamental tool for cancer diagnosis and monitoring, as they reflect tumor heterogeneity and the clonal evolution of cancer in real-time. In particular, EMT phenotypes are commonly associated with therapy resistance. Thus, capturing these CTCs is expected to reveal important clinical information. However, currently available CTC isolation approaches are suboptimal and are often targeted to capture epithelial CTCs, leading to the loss of EMT or mesenchymal CTCs. Here, we describe size-based CTCs isolation using the RUBYchip™, a label-free microfluidic device, aiming to detect EMT biomarkers in CTCs from whole blood samples of GI cancer patients. We found that, for most cases, the mesenchymal phenotype was predominant, and in fact a considerable fraction of isolated CTCs did not express epithelial markers. The RUBYchip™ can overcome the limitations of label-dependent technologies and improve the identification of CTC subpopulations that may be related to different clinical outcomes.

SERS chiral recognition and quantification of enantiomers through cyclodextrin supramolecular complexation.

We introduce here a simple approach in which a cyclodextrin, functionalized with thiols in the narrower rim, is assembled onto the silver surface of a SERS platform composed of polystyrene beads coated with silver nanoparticles. Trapping properties of the fabricated sensor are demonstrated through the retention of different enantiomers (R,R or/and S,S) of hydrobenzoin (HBZ), a molecule that has not been reported before in SERS because it has no affinity for coinage metal surfaces. Further, selective recognition of each enantiomer and semiquantification of its proportion in a racemic mixture are provided by the analysis of the SERS spectra of the HBZ-cyclodextrin complex, in full agreement with the surface selection rules.

Multifunctional microgel magnetic/optical traps for SERS ultradetection.

We report on the fabrication of a SERS substrate comprising magnetic and silver particles encapsulated within a poly(N-isopropylacrylamide) (pNIPAM) thermoresponsive microgel. This colloidal substrate has the ability to adsorb analytes from solution while it is expanded (low temperature) and reversibly generate hot spots upon collapse (high temperature or drying). Additionally, the magnetic functionality permits concentration of the composite particles into small spatial regions, which can be exploited to decrease the amount of material per analysis while improving its SERS detection limit. Proof of concept for the sequestration of uncommon molecular systems is demonstrated through the first SERS analysis of pentachlorophenol (PCP), a chlorinated ubiquitous environmental pollutant.

Use of some cost-effective technologies for a routine clinical pathology laboratory.

Classically, the need for highly sophisticated instruments with important economic costs has been a major limiting factor for clinical pathology laboratories, especially in developing countries. With the aim of making clinical pathology more accessible, a wide variety of free or economical technologies have been developed worldwide in the last few years. 3D printing and Arduino approaches can provide up to 94% economical savings in hardware and instrumentation in comparison to commercial alternatives. The vast selection of point-of-care-tests (POCT) currently available also limits the need for specific instruments or personnel, as they can be used almost anywhere and by anyone. Lastly, there are dozens of free and libre digital tools available in health informatics. This review provides an overview of the state-of-the-art on cost-effective alternatives with applications in routine clinical pathology laboratories. In this context, a variety of technologies including 3D printing and Arduino, lateral flow assays, plasmonic biosensors, and microfluidics, as well as laboratory information systems, are discussed. This review aims to serve as an introduction to different technologies that can make clinical pathology more accessible and, therefore, contribute to achieve universal health coverage.

HER2 Expression in Circulating Tumour Cells Isolated from Metastatic Breast Cancer Patients Using a Size-Based Microfluidic Device.

HER2 is a prognostic and predictive biomarker in breast cancer, normally assessed in tumour biopsy and used to guide treatment choices. Circulating tumour cells (CTCs) escape the primary tumour and enter the bloodstream, exhibiting great metastatic potential and representing a real-time snapshot of the tumour burden. Liquid biopsy offers the unique opportunity for low invasive sampling in cancer patients and holds the potential to provide valuable information for the clinical management of cancer patients. This study assesses the performance of the RUBYchip™, a microfluidic system for CTC capture based on cell size and deformability, and compares it with the only FDA-approved technology for CTC enumeration, CellSearch®. After optimising device performance, 30 whole blood samples from metastatic breast cancer patients were processed with both technologies. The expression of HER2 was assessed in isolated CTCs and compared to tissue biopsy. Results show that the RUBYchipTM was able to isolate CTCs with higher efficiency than CellSearch®, up to 10 times more, averaging all samples. An accurate evaluation of different CTC subpopulations, including HER2+ CTCs, was provided. Liquid biopsy through the use of the RUBYchipTM in the clinic can overcome the limitations of histological testing and evaluate HER2 status in patients in real-time, helping to tailor treatment during disease evolution.

Multifuntional Gold Nanoparticles for the SERS Detection of Pathogens Combined with a LAMP-in-Microdroplets Approach.

We developed a droplet-based optofluidic system for the detection of foodborne pathogens. Specifically, the loop-mediated isothermal amplification (LAMP) technique was combined with surface-enhanced Raman scattering (SERS), which offers an excellent method for DNA ultradetection. However, the direct SERS detection of DNA compromises the simplicity of data interpretation due to the variability of its SERS fingerprints. Therefore, we designed an indirect SERS detection method using multifunctional gold nanoparticles (AuNPs) based on the formation of pyrophosphate generated during the DNA amplification by LAMP. Towards this goal, we prepared multifunctional AuNPs involving three components with key roles: (1) thiolated poly(ethylene glycol) as stabilizing agent, (2) 1-naphthalenethiol as Raman reporter, and (3) glutathione as a bioinspired chelating agent of magnesium (II) ions. Thus, the variation in the SERS signal of 1-naphthalenethiol was controlled by the aggregation of AuNPs triggered by the complexation of pyrophosphate and glutathione with free magnesium ions. Using this strategy, we detected Listeria monocytogenes, not only in buffer, but also in a food matrix (i.e., ultra-high temperaturemilk) enabled by the massive production of hotspots as a result of the self-assemblies that enhanced the SERS signal. This allowed the development of a microdroplet-LAMP-SERS platform with isothermal amplification and real-time identification capabilities.

Loading of exponentially grown LBL films with silver nanoparticles and their application to generalized SERS detection.

Feature film: Thin films made by exponential layer-by-layer growth display high diffusivity and can be readily infiltrated with inorganic nanoparticles. They can sequestrate molecular systems from solution as a function of the composition of their layers, while providing intense surface-enhanced Raman scattering (SERS) signals (see picture).

The Significance of Circulating Tumour Cells in the Clinic.

BACKGROUND: Despite the hype about circulating tumour cells (CTCs) in the early 2000s and their potential in the diagnosis of metastasis, in recent years, the hope for personalised cancer management relies more on circulating tumour (ct)DNA that has entered the clinic in a much more efficient way. So far, approved methods for CTCs in the clinic only provide the counting of CTCs, which enables monitoring of the progression of metastatic breast, prostate, and colorectal cancer patients with therapy. Approved methods for ctDNA facilitate the analysis of specific mutations in lung cancer, thereby providing indications for potentially successful treatments. This situation inclined the balance towards molecular analysis in liquid biopsy, leveraged by new technologies and companies providing broader mutation and gene expression analysis towards the early diagnosis of cancer. STUDY DESIGN: We conducted a search for the studies published to date that provide details about the significance of CTCs in the clinic. RESULTS: Many studies and clinical trials have demonstrated the potential of CTCs in patient screening, early diagnosis, therapy resistance, and patient prognosis. CONCLUSIONS: Large multi-centre studies are still needed to formally validate the clinical relevance of CTCs. Meticulous design of the clinical trials is a crucial point to achieve this long-sought objective.

Amplification-free SERS analysis of DNA mutation in cancer cells with single-base sensitivity.

Accurate and sensitive identification of DNA mutations in tumor cells is critical to the diagnosis, prognosis and personalized therapy of cancer. Conventional polymerase chain reaction (PCR)-based methods are limited by the complicated amplification process. Herein, an amplification-free surface enhanced Raman spectroscopy (SERS) approach which directly detects point mutations in cancer cells has been proposed. A highly sensitive and uniform SERS substrate was fabricated using gold@silver core-shell nanorods, achieving an enhancement factor of 1.85 × 106. By combining the SERS-active nanosubstrate with molecular beacon probes, the limit of detection reached as low as 50 fM. To enable parallel analysis and automated operation, the SERS sensor was integrated into a microfluidic chip. This novel chip-based assay was able to differentiate between mutated and wild-type KRAS genes among a variety of other nucleic acids from cancer cells in 40 min. Owing to the simple operation and fast analysis, the SERS-based DNA assay chip could potentially provide insights into clinical cancer theranostics in an easy and inexpensive manner at the point of care.

Surface-Enhanced Raman Scattering Spectroscopy and Microfluidics: Towards Ultrasensitive Label-Free Sensing.

Raman scattering and surface-enhanced Raman scattering (SERS) spectroscopy have demonstrated their potential as ultrasensitive detection techniques in the past decades. Specifically, and as a result of the flourishing of nanotechnology, SERS is nowadays one of the most powerful sensing techniques, not only because of the low detection limits that it can achieve, but also for the structural information that it offers and its capability of multiplexing. Similarly, microfluidics technology is having an increased presence not only in fundamental research, but also in the industry. The latter is because of the intrinsic characteristics of microfluidics, being automation, high-throughput, and miniaturization. However, despite miniaturization being an advantage, it comes together with the need to use ultrasensitive techniques for the interrogation of events happening in extremely small volumes. The combination of SERS with microfluidics can overcome bottlenecks present in both technologies. As a consequence, the integration of Raman and SERS in microfluidics is being investigated for the label-free biosensing of relevant research challenges.

Droplet microfluidics for the highly controlled synthesis of branched gold nanoparticles.

The synthesis of anisotropic metallic nanoparticles (NPs) has been a field of intense and challenging research in the past decade. In this communication, we report on the reproducible and highly controllable synthesis of monodisperse branched gold nanoparticles in a droplet-based microfluidics platform. The process has been automated by adapting two different bulk synthetic strategies to microdroplets, acting as microreactors, for NP synthesis: a surfactant-free synthesis and a surfactant-assisted synthesis. Microdroplets were generated in two different microfluidic devices designed to accommodate the requirements of both bulk syntheses. The epitaxial growth of AuNSTs inside the microdroplets allowed for a fine control of reagent mixing and local concentrations during particle formation. This is the first time branched gold NPs have been synthesised in a microfluidics platform. The monodispersity of the product was comparable to the synthesis in bulk, proving the potential of this technology for the continuous synthesis of high quality anisotropic NPs with improved reproducibility.