Genetic and phenotypic profiling of single living circulating tumor cells from patients with microfluidics.

Accurate prediction of the efficacy of immunotherapy for cancer patients through the characterization of both genetic and phenotypic heterogeneity in individual patient cells holds great promise in informing targeted treatments, and ultimately in improving care pathways and clinical outcomes. Here, we describe the nanoplatform for interrogating living cell host-gene and (micro-)environment (NICHE) relationships, that integrates micro- and nanofluidics to enable highly efficient capture of circulating tumor cells (CTCs) from blood samples. The platform uses a unique nanopore-enhanced electrodelivery system that efficiently and rapidly integrates stable multichannel fluorescence probes into living CTCs for in situ quantification of target gene expression, while on-chip coculturing of CTCs with immune cells allows for the real-time correlative quantification of their phenotypic heterogeneities in response to immune checkpoint inhibitors (ICI). The NICHE microfluidic device provides a unique ability to perform both gene expression and phenotypic analysis on the same single cells in situ, allowing us to generate a predictive index for screening patients who could benefit from ICI. This index, which simultaneously integrates the heterogeneity of single cellular responses for both gene expression and phenotype, was validated by clinically tracing 80 non-small cell lung cancer patients, demonstrating significantly higher AUC (area under the curve) (0.906) than current clinical reference for immunotherapy prediction.

Real-time single-base specific detection of the Haemonchus contortus S168T variant associated with levamisole resistance using loop-primer endonuclease cleavage loop-mediated isothermal amplification.

Haemonchus contortus is a parasitic haematophagous nematode that primarily affects small ruminants and causes significant economic loss to the global livestock industry. Treatment of haemonchosis typically relies on broad-spectrum anthelmintics, resistance to which is an important cause of treatment failure. Resistance to levamisole remains less widespread than to other major anthelmintic classes, prompting the need for more effective and accurate surveillance to maintain its efficacy. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) is a recently developed diagnostic method that facilitates multiplex target detection with single nucleotide polymorphism (SNP) specificity and portable onsite testing. In this study, we designed a new LEC-LAMP assay and applied it to detect the levamisole resistance marker S168T in H. contortus. We explored multiplexing probes for both the resistant S168T and the susceptible S168 alleles in a single-tube assay. We then included a generic probe to detect the acr-8 gene in the multiplex assay, which could facilitate the quantification of both resistance markers and overall genetic material from H. contortus in a single step. Our results showed promising application of these technologies, demonstrating a proof-of-concept assay which is amenable to detection of resistance alleles within the parasite population, with the potential for multiplex detection, and point-of-care application enabled by lateral flow end-point detection. However, further optimisation and validation is necessary.

An isothermal CRISPR- based lateral flow assay for detection of Neisseria meningitidis.

BACKGROUND: Neisseria meningitidis can cause life-threatening meningococcal meningitis and meningococcemia. Old standard microbiological results from CSF/blood cultures are time consuming. This study aimed to combine the sensitivity of loop-mediated isothermal nucleic acid amplification (LAMP) with the specificity of CRISPR/Cas12a cleavage to demonstrate a reliable diagnostic assay for rapid detection of N. meningitidis. METHODS: A total of n = 139 samples were collected from patients with suspected meningococcal disease and were used for evaluation. The extracted DNA was subjected to qualitative real-time PCR, targeting capsular transporter gene (ctrA) of N. meningitidis. LAMP-specific primer pairs, also targeting the ctrA, were designed and the LAMP products were subjected to CRISPR/Cas12 cleavage reaction. the readout was on a lateral flow strip. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of LAMP-CRISPR/Cas was compared with real-time PCR assays. The limit of detection (LOD) was established with serial dilutions of the target N. meningitidis DNA and calculated by Probit regression analysis. RESULTS: Six LAMP assay-specific primers were developed targeting the ctrA gene of N. meningitidis, which is conserved in all meningococcal serogroups. The LAMP primers did not amplify DNA from other bacterial DNA tested, showing 100% specificity. The use of 0.4 M betaine increased the sensitivity and stability of the reaction. LAMP-CRISPR/Cas detected meningococcal serogroups (B, C, W). The assay showed no cross-reactivity and was specific for N. meningitidis. The LOD was 74 (95% CI: 47-311) N. meningitidis copies. The LAMP-CRISPR/Cas performed well compared to the gold standard. In the 139 samples from suspected patients, the sensitivity and specificity of the test were 91% and 99% respectively. CONCLUSION: This developed and optimized method can complement for the available gold standard for the timely diagnosis of meningococcal meningitis and meningococcemia.

Mobile Health Access and Utilisation in Uganda: Knowledge, Attitudes and Perceptions of Health and Veterinary Workers.

Background: Despite its strong growth in many parts of the world, mobile health access is still limited in low- and middle-income countries. Among the many factors restricting implementation are the lack of information security, insufficient evidence base, low sensitization, and user acceptance. Limited evidence has been obtained on current practices, perceptions, and user acceptability in such settings. The aim of this study was therefore to evaluate the knowledge, attitude, and perceptions on mobile health use among health workers and veterinary officers in Uganda. Materials and Methods: A cross-section study was carried out, targeting health practitioners in both hospitals and veterinary laboratories/clinics. A structured questionnaire was used to collect data from the Central, Eastern, Northern, and Western representative regions. Interviews with selected health workers were also conducted as well as a focused group discussion. Results: Of the 120 health practitioners that were targeted, a total of 80 health workers and 7 veterinary practitioners participated in the study of which 46% were men and 54% women. Majority of the health workers had encountered m-health but had never used it, whereas the 15 practitioners who had used it before the survey did not use it for disease diagnosis in hospitals but used it for ordering medicine online, for patient consultations with the doctors, result interpretation, tracking women menstrual cycles, tuberculosis assessment. Discussion and Conclusion: Participants expressed significant interest in mobile health as it addresses key challenges including challenges with management of patient data, and long patient queues, which would ultimately improve service delivery. However, there is some skepticism about access as many rural facilities lack access to smartphones and stable internet.

Changes in Oxygenation Levels During Moderate Altitude Simulation (Hypoxia-Induced): A Pilot Study Investigating the Impact of Skin Pigmentation in Pulse Oximetry.

The current COVID-19 pandemic has shown us that the pulse oximeter is a key medical device for monitoring blood-oxygen levels non-invasively in patients with chronic or acute illness. It has also emphasised limitations in accuracy for individuals with darker skin pigmentation, calling for new methods to provide better measurements. The aim of our study is to identify the impact of skin pigmentation on pulse oximeter measurements. We also explored the benefits of a multi-wavelength approach with an induced change of arterial oxygen saturation. A total of 20 healthy volunteers were recruited. We used time domain diffuse reflectance spectroscopy (TDDRS) from a broad band light source, collecting spectra from the index finger along with three different pulse oximeters used simultaneously for monitoring purposes. Five acute hypoxic events were induced by administering 11% FiO2, produced by a Hypoxico altitude training system, for 120 sec through a face mask with a one-way valve. Our multi-wavelength approach revealed a correlation between the signature of skin pigmentation and the dynamic range of oxygen saturation measurements. Principal component analysis (PCA) showed separation between a range of different pigmented volunteers (PC1 = 56.00%) and oxygen saturation (PC2 = 22.99%). This emphasises the need to take into account skin pigmentation in oximeter measurements. This preliminary study serves to validate the need to better understand the impact of skin pigmentation absorption on optical readings in pulse oximeters. Multi-wavelength approaches have the potential to enable robust and accurate measurements across diverse populations.

Paper-based microfluidics for DNA diagnostics of malaria in low resource underserved rural communities.

Rapid, low-cost, species-specific diagnosis, based upon DNA testing, is becoming important in the treatment of patients with infectious diseases. Here, we demonstrate an innovation that uses origami to enable multiplexed, sensitive assays that rival polymerase chain reactions (PCR) laboratory assays and provide high-quality, fast precision diagnostics for malaria. The paper-based microfluidic technology proposed here combines vertical flow sample-processing steps, including paper folding for whole-blood sample preparation, with an isothermal amplification and a lateral flow detection, incorporating a simple visualization system. Studies were performed in village schools in Uganda with individual diagnoses being completed in <50 min (faster than the standard laboratory-based PCR). The tests, which enabled the diagnosis of malaria species in patients from a finger prick of whole blood, were both highly sensitive and specific, detecting malaria in 98% of infected individuals in a double-blind first-in-human study. Our method was more sensitive than other field-based, benchmark techniques, including optical microscopy and industry standard rapid immunodiagnostic tests, both performed by experienced local healthcare teams (which detected malaria in 86% and 83% of cases, respectively). All assays were independently validated using a real-time double-blinded reference PCR assay. We not only demonstrate that advanced, low-cost DNA-based sensors can be implemented in underserved communities at the point of need but also highlight the challenges associated with developing and implementing new diagnostic technologies in the field, without access to laboratories or infrastructure.

A rapid variant-tolerant reverse transcription loop-mediated isothermal amplification assay for the point of care detection of HIV-1.

Human immunodeficiency virus (HIV) continues to be a major burden on public health globally with on-going increases in the number of new infections each year. Rapid and sensitive point-of-care tests allow timely interventions and are essential to control the spread of the disease. However the highly variable nature of the virus, resulting in the evolution of many subtypes and inter-subtype recombinants, poses important challenges for its diagnosis. Here we describe a variant-tolerant reverse-transcription RT-LAMP amplification of the virus's INT gene, providing a simple to use, rapid (<30 min) in vitro point-of-care diagnostic test with a limit of detection <18 copies/reaction. The assay was first validated in clinical studies of patient samples, using both established RT-LAMP and RT-qPCR assays for reference, with results showing that this new variant-tolerant HIV-1 RT-LAMP diagnostic test is highly sensitive without compromising its high specificity for HIV-1 subtypes. The diagnostic test was subsequently configured within an easy-to-read paper microfluidic lateral flow test and was validated clinically using patient samples, demonstrating its future potential for use in timely, effective, low cost HIV diagnostics in global regions where healthcare resources may be limited.

Molecular clutch drives cell response to surface viscosity.

Cell response to matrix rigidity has been explained by the mechanical properties of the actin-talin-integrin-fibronectin clutch. Here the molecular clutch model is extended to account for cell interactions with purely viscous surfaces (i.e., without an elastic component). Supported lipid bilayers present an idealized and controllable system through which to study this concept. Using lipids of different diffusion coefficients, the mobility (i.e., surface viscosity) of the presented ligands (in this case RGD) was altered by an order of magnitude. Cell size and cytoskeletal organization were proportional to viscosity. Furthermore, there was a higher number of focal adhesions and a higher phosphorylation of FAK on less-mobile (more-viscous) surfaces. Actin retrograde flow, an indicator of the force exerted on surfaces, was also seen to be faster on more mobile surfaces. This has consequential effects on downstream molecules; the mechanosensitive YAP protein localized to the nucleus more on less-mobile (more-viscous) surfaces and differentiation of myoblast cells was enhanced on higher viscosity. This behavior was explained within the framework of the molecular clutch model, with lower viscosity leading to a low force loading rate, preventing the exposure of mechanosensitive proteins, and with a higher viscosity causing a higher force loading rate exposing these sites, activating downstream pathways. Consequently, the understanding of how viscosity (regardless of matrix stiffness) influences cell response adds a further tool to engineer materials that control cell behavior.

Hierarchical Nanotexturing Enables Acoustofluidics on Slippery yet Sticky, Flexible Surfaces.

The ability to actuate liquids remains a fundamental challenge in smart microsystems, such as those for soft robotics, where devices often need to conform to either natural or three-dimensional solid shapes, in various orientations. Here, we propose a hierarchical nanotexturing of piezoelectric films as active microfluidic actuators, exploiting a unique combination of both topographical and chemical properties on flexible surfaces, while also introducing design concepts of shear hydrophobicity and tensile hydrophilicity. In doing so, we create nanostructured surfaces that are, at the same time, both slippery (low in-plane pinning) and sticky (high normal-to-plane liquid adhesion). By enabling fluid transportation on such arbitrarily shaped surfaces, we demonstrate efficient fluid motions on inclined, vertical, inverted, or even flexible geometries in three dimensions. Such surfaces can also be deformed and then reformed into their original shapes, thereby paving the way for advanced microfluidic applications.

Lipid topology and electrostatic interactions underpin lytic activity of linear cationic antimicrobial peptides in membranes.

Linear cationic antimicrobial peptides are a diverse class of molecules that interact with a wide range of cell membranes. Many of these peptides disrupt cell integrity by forming membrane-spanning pores that ultimately lead to their death. Despite these peptides high potency and ability to evade acquired bacterial drug resistance, there is a lack of knowledge on their selectivity and activity mechanisms. Such an understanding would provide an informative framework for rational design and could lead to potential antimicrobial therapeutic targets. In this paper, we use a high-throughput microfluidic platform as a quantitative screen to assess peptide activity and selectivity by precisely controlling exposure to vesicles with lipid compositions that mimic both bacterial and mammalian cell membranes. We explore the complexity of the lipid-peptide interactions governing membrane-disruptive behaviors and establish a link between peptide pore formation and both lipid-peptide charge and topological interactions. We propose a topological model for linear antimicrobial peptide activity based on the increase in membrane strain caused by the continuous adsorption of peptides to the target vesicle coupled with the effects of both lipid-peptide charge and topographical interactions. We also show the validity of the proposed model by investigating the activity of two prototypical linear cationic peptides: magainin 2 amide (which is selective for bacterial cells) and melittin (which targets both mammalian and bacterial cells indiscriminately). Finally, we propose the existence of a negative feedback mechanism that governs the pore formation process and controls the membrane's apparent permeability.

Breaking the Symmetry of Momentum Conservation Using Evanescent Acoustic Fields.

Although the conservation of momentum is a fundamental law in physics, its constraints are not fulfilled for wave propagation at material boundaries, where incident waves give rise to evanescent field distributions. While nonlinear susceptibility tensor terms can provide solutions in the optical regime, this framework cannot be applied directly to acoustic waves. Now, by considering a complete representation of wave interactions and scattering at boundaries, we are able to show a generic formalism of sum-frequency mixing for the whole scattering field including all evanescent waves. This general case was studied analytically and verified both numerically and experimentally for ultrasonic waves, showing that considering evanescent waves leads to an anomalous nonlinear interaction which enhances sum-frequency generation. This new interpretation not only provides a deeper understanding of the momentum conservation laws in acoustics but also promises translation of this new understanding into optics and photonics, to enhance nonlinear interactions.

Monitoring Genetic Population Biomarkers for Wastewater-Based Epidemiology.

We report a rapid "sample-to-answer" platform that can be used for the quantitative monitoring of genetic biomarkers within communities through the analysis of wastewater. The assay is based on the loop-mediated isothermal amplification (LAMP) of nucleic acid biomarkers and shows for the first time the ability to rapidly quantify human-specific mitochondrial DNA (mtDNA) from raw untreated wastewater samples. mtDNA provides a model population biomarker associated with carcinogenesis including breast, renal and gastric cancers. To enable a sample-to-answer, field-based technology, we integrated a filter to remove solid impurities and perform DNA extraction and enrichment into a low cost lateral flow-based test. We demonstrated mtDNA detection over seven consecutive days, achieving a limit of detection of 40 copies of human genomic DNA per reaction volume. The assay can be performed at the site of sample collection, with minimal user intervention, yielding results within 45 min and providing a method to monitor public health from wastewater.

Lateral Chain Length in Polyalkyl Acrylates Determines the Mobility of Fibronectin at the Cell/Material Interface.

Cells, by interacting with surfaces indirectly through a layer of extracellular matrix proteins, can respond to a variety of physical properties, such as topography or stiffness. Polymer surface mobility is another physical property that is less well understood but has been indicated to hold the potential to modulate cell behavior. Polymer mobility is related to the glass-transition temperature (Tg) of the system, the point at which a polymer transitions from an amorphous solid to a more liquid-like state. This work shows that changes in polymer mobility translate to interfacial mobility of extracellular matrix proteins adsorbed on the material surface. This study has utilized a family of polyalkyl acrylates with similar chemistry but different degrees of mobility, obtained through increasing length of the side chain. These materials are used, in conjunction with fluorescent fibronectin, to determine the mobility of this interfacial layer of protein that constitutes the initial cell-material interface. Furthermore, the extent of fibronectin domain availability (III9, III10, - the integrin binding site), cell-mediated reorganization, and cell differentiation was also determined. A nonmonotonic dependence of fibronectin mobility on polymer surface mobility was observed, with a similar trend noted in cell-mediated reorganization of the protein layer by L929 fibroblasts. The availability of the integrin-binding site was higher on the more mobile surfaces, where a similar organization of the protein into networks at the material interface was observed. Finally, differentiation of C2C12 myoblasts was seen to be highly sensitive to surface mobility upon inhibition of cell contractility. Altogether, these findings show that polymer mobility is a subtle influence that translates to the cell/material interface through the protein layer to alter the biological activity of the surface.

Paper-Origami-Based Multiplexed Malaria Diagnostics from Whole Blood.

We demonstrate, for the first time, the multiplexed determination of microbial species from whole blood using the paper-folding technique of origami to enable the sequential steps of DNA extraction, loop-mediated isothermal amplification (LAMP), and array-based fluorescence detection. A low-cost handheld flashlight reveals the presence of the final DNA amplicon to the naked eye, providing a "sample-to-answer" diagnosis from a finger-prick volume of human blood, within 45 min, with minimal user intervention. To demonstrate the method, we showed the identification of three species of Plasmodium, analyzing 80 patient samples benchmarked against the gold-standard polymerase chain reaction (PCR) assay in an operator-blinded study. We also show that the test retains its diagnostic accuracy when using stored or fixed reference samples.

Chemical-free lysis and fractionation of cells by use of surface acoustic waves for sensitive protein assays.

We exploit the mechanical action of surface acoustic waves (SAW) to differentially lyse human cancer cells in a chemical-free manner. The extent to which cells were disrupted is reported for a range of SAW parameters, and we show that the presence of 10 µm polystyrene beads is required to fully rupture cells and their nuclei. We show that SAW is capable of subcellular fractionation through the chemical-free isolation of nuclei from whole cells. The concentration of protein was assessed in lysates with a sensitive microfluidic antibody capture (MAC) chip. An antibody-based sandwich assay in a microfluidic microarray format was used to detect unlabeled human tumor suppressor protein p53 in crude lysates, without any purification step, with single-molecule resolution. The results are digital, enabling sensitive quantification of proteins with a dynamic range >4 orders of magnitude. For the conditions used, the efficiency of SAW-induced mechanical lysis was determined to be 12.9% ± 0.7% of that for conventional detergent-based lysis in yielding detectable protein. A range of possible loss mechanisms that could lead to the drop in protein yield are discussed. Our results show that the methods described here are amenable to an integrated point-of-care device for the assessment of tumor protein expression in fine needle aspirate biopsies.

Acoustic suppression of the coffee-ring effect.

We study the influence of acoustic fields on the evaporative self-assembly of solute particles suspended inside sessile droplets of complex fluids. The self-assembly process often results in an undesirable ring-like heterogeneous residue, a phenomenon known as the coffee-ring effect. Here we show that this ring-like self-assembly can be controlled acoustically to form homogeneous disc-like or concentrated spot-like residues. The principle of our method lies in the formation of dynamic patterns of particles in acoustically excited droplets, which inhibits the evaporation-driven convective transport of particles towards the contact line. We elucidate the mechanisms of this pattern formation and also obtain conditions for the suppression of the coffee-ring effect. Our results provide a more general solution to suppress the coffee-ring effect without any physiochemical modification of the fluids, the particles or the surface, thus potentially useful in a broad range of industrial and analytical applications that require homogenous solute depositions.

Shaping acoustic fields as a toolset for microfluidic manipulations in diagnostic technologies.

Ultrasonics offers the possibility of developing sophisticated fluid manipulation tools in lab-on-a-chip technologies. Here we demonstrate the ability to shape ultrasonic fields by using phononic lattices, patterned on a disposable chip, to carry out the complex sequence of fluidic manipulations required to detect the rodent malaria parasite Plasmodium berghei in blood. To illustrate the different tools that are available to us, we used acoustic fields to produce the required rotational vortices that mechanically lyse both the red blood cells and the parasitic cells present in a drop of blood. This procedure was followed by the amplification of parasitic genomic sequences using different acoustic fields and frequencies to heat the sample and perform a real-time PCR amplification. The system does not require the use of lytic reagents nor enrichment steps, making it suitable for further integration into lab-on-a-chip point-of-care devices. This acoustic sample preparation and PCR enables us to detect ca. 30 parasites in a microliter-sized blood sample, which is the same order of magnitude in sensitivity as lab-based PCR tests. Unlike other lab-on-a-chip methods, where the sample moves through channels, here we use our ability to shape the acoustic fields in a frequency-dependent manner to provide different analytical functions. The methods also provide a clear route toward the integration of PCR to detect pathogens in a single handheld system.

Spatially selecting a single cell for lysis using light-induced electric fields.

An optoelectronic tweezing (OET) device, within an integrated microfluidic channel, is used to precisely select single cells for lysis among dense populations. Cells to be lysed are exposed to higher electrical fields than their neighbours by illuminating a photoconductive film underneath them. Using beam spot sizes as low as 2.5 µm, 100% lysis efficiency is reached in <1 min allowing the targeted lysis of cells.

Rare-cell enrichment by a rapid, label-free, ultrasonic isopycnic technique for medical diagnostics.

One significant challenge in medical diagnostics lies in the development of label-free methods to separate different cells within complex biological samples. Here we demonstrate a generic, low-power ultrasonic separation technique, able to enrich different cell types based upon their physical properties. For malaria, we differentiate between infected and non-infected red blood cells in a fingerprick-sized drop of blood. We are able to achieve an enrichment of circulating cells infected by the ring stage of the parasite over nonparasitized red blood cells by between two and three orders of magnitude in less than 3 seconds (enabling detection at parasitemia levels as low as 0.0005%). In a second example, we also show that our methods can be used to enrich different cell types, concentrating Trypanosoma in blood at very low levels of infection, on disposable, low-cost chips.

Shape-dependent optoelectronic cell lysis.

We show an electrical method to break open living cells amongst a population of different cell types, where cell selection is based upon their shape. We implement the technique on an optoelectronic platform, where light, focused onto a semiconductor surface from a video projector creates a reconfigurable pattern of electrodes. One can choose the area of cells to be lysed in real-time, from single cells to large areas, simply by redrawing the projected pattern. We show that the method, based on the "electrical shadow" that the cell casts, allows the detection of rare cell types in blood (including sleeping sickness parasites), and has the potential to enable single cell studies for advanced molecular diagnostics, as well as wider applications in analytical chemistry.