Microfluidic Sample Research Articles

A non-invasive wearable stress patch for real-time cortisol monitoring using a pseudoknot-assisted aptamer

Stress is part of everyone's life and is exacerbated by traumatic events such as pandemics, disasters, violence, lifestyle changes, and health disorders. Chronic stress has many detrimental health effects and can even be life-threatening. Long-term stress monitoring outside of a hospital is often accomplished by measuring heart rate variability. While easy to measure, this digital biomarker has low specificity, greatly limiting its utility. To address this shortcoming, we report a non-invasive, wearable biomolecular sensor to monitor cortisol levels in sweat. Cortisol is a neuroendocrine hormone that regulates homeostasis as part of the stress pathway. Cortisol is detected using an electrochemical sensor functionalized with a pseudoknot-assisted aptamer and a flexible microfluidic sweat sampling system. The skin-worn microfluidic sampler provides rapid sweat collection while separating old and new sweat. The conformation-switching aptamer provides high specificity towards cortisol while being regenerable, allowing it to monitor temporal changes continuously. The aptamer was engineered to add a pseudoknot, restricting it to only two states, thus minimizing the background signal and enabling high sensitivity. An electrochemical pH sensor allows pH-corrected amperometric measurements. Device operation was demonstrated invitro with a broad linear dynamic range (1 pM - 1 μM) covering the physiological range and a sub-picomolar (0.2 pM) limit of detection in sweat. Real-time, on-body measurements were collected from human subjects using an induced stress protocol, demonstrating in-situ signal regeneration and the ability to detect dynamic cortisol fluctuations continuously for up to 90 min. The reported device has the potential to improve prognosis and enable personalized treatments.

Multifunctional acoustofluidic centrifuge device using tri-symmetrical design for particle enrichment and separation and multiphase microflow mixing

Effective enrichment and separation of particles and mixing of multiphase microflows can be technically challenging in microfluidic sample processing and analysis. Acoustofluidic techniques have advantages such as high biocompatibility, versatility, and simple device design to meet these demands. Here, we present a novel tri-symmetrical acoustofluidic centrifuge based on traveling surface acoustic waves (TSAWs), achieving particle enrichment and bidirectional size-based separation, pushing away the larger particles while enriching the smaller ones, in a rapid time (<6 s). A microfluidic chamber was designed for further extraction of treated particles and cells. Experimental results show that our device can achieve enrichment of particles in a range of sizes and high purity separation (e.g. >90 % in 5 µm and 40 µm sample), demonstrating its sample processing capabilities. The acoustic treatment did not affect cells (consistent with the control group above 80 %). TSAW-induced acoustic streaming enabled efficient mixing (mixing efficiency χ >85 %) of fluorescent multi-phase microflows within a short time (<0.5 s). The underlying physical mechanisms have been verified by numerical simulations.

Manufacturing of Ultra-Thin X-ray-Compatible COC Microfluidic Devices for Optimal In Situ Macromolecular Crystallography Experiments.

Cyclic-olefin-copolymer (COC)-based microfluidic devices are increasingly becoming the center of highly valuable research for in situ X-ray measurements due to their compatibility with X-rays, biological compounds, chemical resistance, optical properties, low cost, and simplified handling. COC microfluidic devices present potential solutions to challenging biological applications such as protein binding, folding, nucleation, growth kinetics, and structural changes. In recent years, the techniques applied to manufacturing and handling these devices have capitalized on enormous progress toward small-scale sample probing. Here, we describe the new and innovative design aspects, fabrication, and experimental implementation of low-cost and micron-sized X-ray-compatible microfluidic sample environments that address diffusion-based crystal formation for crystallographic characterization. The devices appear fully compatible with crystal growth and subsequent X-ray diffraction experiments, resulting in remarkably low background data recording. The results highlighted in this research demonstrate how the engineered microfluidic devices allow the recording of accurate crystallographic data at room temperature and structure determination at high resolution.

Concepts for millimeter wave-based detection of African trypanosomes in field-compatible liquid systems

Abstract Human African Trypanosomiasis (HAT) is caused by the African trypanosome, a single-cell parasite that proliferates in the blood and cerebrospinal fluid of infected patients. Diagnostic measures for this pathogen are currently not sufficiently robust and reliable enough to permit effective disease control procedures. As a consequence, we suggested the development of a new sensor type, combining the selectivity of parasite-specific nucleic acid aptamers with the sensitivity of resonant electromagnetic devices to capture and detect the disease-causing organism. While we accomplished the detection of parasite cells in dehydrated specimens, here we summarize our recent progress toward electromagnetic sensors capable of uncovering parasites in liquid patient samples. We present a technique for the removal of blood cells from blood specimens and the deposition of trypanosome cells on glass microfiber membranes for dielectric spectrometry. Liquid suspensions of trypanosomes are characterized to determine the actual dielectric properties of single parasites and lastly, we present two sensor concepts optimized for the detection in liquids, along with a fabrication technique for the integration of microfluidic sample confinements.

A wearable electrochemical biosensor for the monitoring of metabolites and nutrients.

Wearable non-invasive biosensors for the continuous monitoring of metabolites in sweat can detect a few analytes at sufficiently high concentrations, typically during vigorous exercise so as to generate sufficient quantity of the biofluid. Here we report the design and performance of a wearable electrochemical biosensor for the continuous analysis, in sweat during physical exercise and at rest, of trace levels of multiple metabolites and nutrients, including all essential amino acids and vitamins. The biosensor consists of graphene electrodes that can be repeatedly regenerated in situ, functionalized with metabolite-specific antibody-like molecularly imprinted polymers and redox-active reporter nanoparticles, and integrated with modules for iontophoresis-based sweat induction, microfluidic sweat sampling, signal processing and calibration, and wireless communication. In volunteers, the biosensor enabled the real-time monitoring of the intake of amino acids and their levels during physical exercise, as well as the assessment of the risk of metabolic syndrome (by correlating amino acid levels in serum and sweat). The monitoring of metabolites for the early identification of abnormal health conditions could facilitate applications in precision nutrition.

Red blood cell distribution width is associated with increased interactions of blood cells with vascular wall

The mechanism underlying the association between elevated red cell distribution width (RDW) and poor prognosis in variety of diseases is unknown although many researchers consider RDW a marker of inflammation. We hypothesized that RDW directly affects intravascular hemodynamics, interactions between circulating cells and vessel wall, inducing local changes predisposing to atherothrombosis. We applied different human and animal models to verify our hypothesis. Carotid plaques harvested from patients with high RDW had increased expression of genes and proteins associated with accelerated atherosclerosis as compared to subjects with low RDW. In microfluidic channels samples of blood from high RDW subjects showed flow pattern facilitating direct interaction with vessel wall. Flow pattern was also dependent on RDW value in mouse carotid arteries analyzed with Magnetic Resonance Imaging. In different mouse models of elevated RDW accelerated development of atherosclerotic lesions in aortas was observed. Therefore, comprehensive biological, fluid physics and optics studies showed that variation of red blood cells size measured by RDW results in increased interactions between vascular wall and circulating morphotic elements which contribute to vascular pathology.

Twisted light Michelson interferometer for high precision refractive index measurements.

Using orbital angular momentum beams in a Michelson interferometer opens the possibility for non-invasive measurements of refractive index changes down to 10-6 refractive index units. We demonstrate the application of a twisted light interferometer to directly measure the concentration of NaCl and glucose solutions label-free and in situ and to monitor temperature differences in the mK-µK range. From these measurements we can extract a correlation of the refractive index to concentration and to temperature from a liquid sample which is in good agreement with literature. Applying this type of twisted light interferometry yields a novel, robust, and easily implementable method for in situ monitoring of concentration and temperature changes in microfluidic samples.

P-084 Novel Single-Step Microfluidic Chamber Outperforms Density Gradient Centrifugation in Post-Selection Motility And DNA Integrity

Abstract Study question Does sperm selection by a novel single-step microfluidic chamber offer effective selection of motile sperm without DNA damage compared with Density Gradient Centrifugation (DGC)? Summary answer The novel single-step microfluidic chamber is a good alternative to conventional DGC, increasing DNA integrity and progressive sperm motility in recovered sperm populations. What is known already High levels of sperm DNA fragmentation (SDF) can have negative impacts on reproductive outcomes such as clinical pregnancy and miscarriage rates. Microfluidic sperm selection technologies have been developed to select sperm with lower level of SDF and higher levels of motility to improve Assisted Reproductive technology (ART) outcomes. While these methods have achieved variable success, very few have proven clinically relevant and fail to perform selection in a robust and simple methodology that can be adopted in clinics. Study design, size, duration A prospective cohort study including 21 donated semen samples processed in 2021. Each sample was split to perform DGC and microfluidic sperm selections side-by-side. Laboratory outcomes were evaluated using semen parameters pre and post processing and included concentration, motility, and DNA fragmentation index (DFI). Participants/materials, setting, methods The study was performed using samples donated at the Department of Obstetrics and Gynaecology, Monash University, Melbourne, Australia. DGC (10 minutes at 300g and 5 minutes wash at 300g) and microfluidic sperm selection (one-step sperm selection;15-minute incubation for selection) were performed with Sydney IVF 80/40 gradients and gamete buffers, respectively. DFI was assessed by chromatin sperm dispersion assay (SCD). Results were analysed by Friedman’s test and differences were considered significant when p-value &amp;lt; 0.05. Main results and the role of chance For the unprocessed raw semen, the starting DFI values varied from 3.0 to 23.3% with an average of 11.38% ± 5.51 and motility varied between 25.3% and 86.7% with an average of 56.42% ± 18.57. The device consistently outperformed DGC in all 21 samples for DFI (0.94% ± 1.12 vs 4.87% ±3.69, P = 0.0012) and in 20 of the 21 samples for motility post-processing DGC (90.88% ± 6.8 vs 73.58% ± 14.97, P = 0.0061). The average concentrations of the raw, DGC, and microfluidic sperm samples were 94.5, 74.12, and 3.55 million sperm/mL, respectively. Thus, both DGC and the microfluidic device showed improvements in SDF, motility, and concentration over the raw sample. However, the microfluidic device significantly outperformed DGC in terms of percentage SDF and motility, indicating its potential for clinical implementation. Limitations, reasons for caution This cohort study did not include samples from infertile patients. Furthermore, the effect of each sperm selection methods on reproductive outcomes was not a part of the study. A larger number of samples across a range of clinically infertile samples is required to fully characterise the microfluidic devices efficacy. Wider implications of the findings The microfluidic device we report here has the potential to become a new tool to improve the efficacy and consistency of sperm selections. When compared to DGC it offers improved sperm motility and DNA integrity from a platform that is simple to use and less skill-based than conventional methods. Trial registration number not applicable

Microfluidic Sampling and Biosensing Systems for Foodborne Escherichia coli and Salmonella.

Developments of portable biosensors for field-deployable detections have been increasingly important to control foodborne pathogens in regulatory environment and in early stage of outbreaks. Conventional cultivation and gene amplification methods require sophisticated instruments and highly skilled professionals; while portable biosensing devices provide more freedom for rapid detections not only in research laboratories but also in the field; however, their sensitivity and specificity are limited. Microfluidic methods have the advantage of miniaturizing instrumental size while integrating multiple functions and high-throughput capability into one streamlined system at low cost. Minimal sample consumption is another advantage to detect samples in different sizes and concentrations, which is important for the close monitoring of pathogens at consumer end. They improve measurement or manipulation of bacteria by increasing the ratio of functional interface of the device to the targeted biospecies and in turn reducing background interference. This article introduces the major active and passive microfluidic devices that have been used for bacteria sampling and biosensing. The emphasis is on particle-based sorting/enrichment methods with or without external physical fields applied to the microfluidic devices and on various biosensing applications reported for bacteria sampling. Three major fabrication methods for microfluidics are briefly discussed with their advantages and limitations. The applications of these active and passive microfluidic sampling methods in the past 5 years have been summarized, with the focus on Escherichia coli and Salmonella. The current challenges to microfluidic bacteria sampling are caused by the small size and nonspherical shape of various bacterial cells, which can induce unpredictable deviations in sampling and biosensing processes. Future studies are needed to develop rapid prototyping methods for device manufacturing, which can facilitate rapid response to various foodborne pathogen outbreaks.

Microfluidic device integrating single-cell extraction and electrical lysis for mass spectrometry detection of intracellular compounds.

Analysis of cellular composition and metabolism at a single-cell resolution allows gaining more information about complex relationships of cells within tissues or whole living organisms by resolving the variance stemming from the cellular heterogeneity. Mass spectrometry (MS) is a perfect analytical tool satisfying the demanding requirements of detecting and identifying compounds present in such ultralow-volume samples of high chemical complexity. However, the method of sampling and sample ionization is crucial in obtaining relevant information. In this work, we present a microfluidic sampling platform that integrates single-cell extraction from MS-incompatible media with electrical cell lysis and nanoESI-MS analysis of human erythrocytes. Hemoglobin alpha and beta chains (300amol/cell) were successfully identified in mass spectra of single-erythrocyte lysates.

Three-dimensional feature matching improves coverage for single-cell proteomics based on ion mobility filtering.

SUMMARYSingle-cell proteomics (scProteomics) promises to advance our understanding of cell functions within complex biological systems. However, a major challenge of current methods is their inability to identify and provide accurate quantitative information for low-abundance proteins. Herein, we describe an ion-mobility-enhanced mass spectrometry acquisition and peptide identification method, transferring identification based on FAIMS filtering (TIFF), to improve the sensitivity and accuracy of label-free scProteomics. TIFF extends the ion accumulation times for peptide ions by filtering out singly charged ions. The peptide identities are assigned by a three-dimensional MS1 feature matching approach (retention time, accurate mass, and FAIMS compensation voltage). The TIFF method enabled unbiased proteome analysis to a depth of >1,700 proteins in single HeLa cells, with >1,100 proteins consistently identified. As a demonstration, we applied the TIFF method to obtain temporal proteome profiles of >150 single murine macrophage cells during lipopolysaccharide stimulation and identified time-dependent proteome changes. A record of this paper’s transparent peer review process is included in the supplemental information.

Microfluidic volume optical monitoring system based on functional channels integrated by hollow cylindrical waveguide

Real-time monitoring and manipulating various liquids in the microflow channels have great potentials in micro total analysis system. A novel microfluidics volume optical monitoring system (MVOMS) is proposed. Functional channels embedded with hollow cylindrical waveguide (HCW) are integrated in MVOMS, which is convenient to realize the real-time monitoring and manipulating microfluidic. The microfluidic volume can be monitored in real time and the sensitivity can reach 20.0 dB/μL. In addition, the MVOMS can realize the monitoring of various liquid for the designed sensor structure is insensitive to the refractive index of liquid. The temperature cross and the pressure cross are tested that can be ignored. Microfluidic volume monitoring, delivering and mixing can be achieved in MVOMS. On this basis, we design an integrated light-driven microfluidic quantitative sampling system, which can sample a specific volume of microfluidic in microchips automatically and accurately.

Point-of-care therapeutic drug monitoring of adalimumab by integrating a FO-SPR biosensor in a self-powered microfluidic cartridge

Disease treatment with advanced biological therapies such as adalimumab (ADM), although largely beneficial, is still costly and suffers from loss of response. To tackle these aspects, therapeutic drug monitoring (TDM) is proposed to improve treatment dosing and efficacy, but is often associated with long sampling-to-result workflows. Here, we present an in-house constructed ADM-sensor, allowing TDM of ADM at the doctor's office. This biosensor brings fiber optic surface plasmon resonance (FO-SPR), combined with self-powered microfluidics, to a point of care (POC) setting for the first time. After developing a rapid FO-SPR sandwich bioassay for ADM detection on a commercial FO-SPR device, this bioassay was implemented on the fully-integrated ADM-sensor. For the latter, we combined (I) a gold coated fiber optic (FO) probe for bioassay implementation and (II) an FO-SPR readout system with (III) the self-powered iSIMPLE microfluidic technology empowering plasma sample and reagent mixing on the-cartridge as well as connection to the FO-SPR readout system. With a calculated limit of detection (LOD) of 0.35 μg/mL in undiluted plasma, and a total time-to-result (TTR) within 12 min, this innovative biosensor demonstrated a comparable performance to existing POC biosensors for ADM quantification in patient plasma samples, while requiring only 1 μL of plasma. Whereas this study demonstrates great potential for FO-SPR biosensing at the POC using ADM as a model case, it also shows huge potential for bedside TDM of other drugs (e.g. other immunosuppressants, anti-epileptics and antibiotics), as the bioassay is highly amenable to adaptation.

Implementation of wedged-serial protein crystallography at PROXIMA-1.

An approach for serial crystallography experiments based on wedged-data collection is described. This is an alternative method for recording in situ X-ray diffraction data on crystalline samples efficiently loaded in an X-ray compatible microfluidic chip. Proper handling of the microfluidic chip places crystalline samples at geometrically known positions with respect to the focused X-ray interaction area for serial data collection of small wedges. The integration of this strategy takes advantage of the greatly modular sample environment available on the endstation, which allows access to both in situ and more classical cryo-crystallography with minimum time loss. The method represents another optional data collection approach that adds up to the already large set of methods made available to users. Coupled with the advances in processing serial crystallography data, the wedged-data collection strategy proves highly efficient in minimizing the amount of required sample crystals for recording a complete dataset. From the advances in microfluidic technology presented here, high-throughput room-temperature crystallography experiments may become routine and should be easily extended to industrial use.

Non-equilibrium potentiometric sensors integrated with metal modified paper-based microfluidic solution sampling substrates for determination of heavy metals in complex environmental samples

Metal modified paper-based substrates were utilized for microfluidic paper-based solution sampling coupled with Pb2+-ion selective electrodes (ISEs) with the aim of controlling the super-Nernstian response which usually occurs when using unmodified paper substrates. Potentiometric responses of Pb2+-ISEs coupled with gold, platinum and palladium coated paper substrates were investigated. Potentiometric response time was found to be predominantly dependent on the thickness of metallic layer deposited at the paper substrates. Paper-based substrates coated on both sides with 38 nm gold layers were found to be the most advantageous in controlling the super-Nernstian response of ISEs at non-equilibrium conditions. Durability studies indicated that the lifetime of Pb2+-ISEs could be doubled when used with paper-based substrates in complex environmental samples with high solid-to-liquid content. Determination of lead in real samples using metal modified paper substrates coupled with Pb2+-ISEs was validated by inductively coupled plasma optical emission spectrometry (ICP-OES). Detailed life cycle assessments were performed for model screen printed potentiometric sensors with and without metal modified paper-based solution sampling substrates. The results confirmed that the use of modified paper substrates demonstrated lower environmental impact per potentiometric measurement of Pb2+-ISE in prepared simulated environmental sample as compared to sensors without the use of paper substrates.

Ion-selective membrane modified microfluidic paper-based solution sampling substrates for potentiometric heavy metal detection.

Paper-based microfluidic solution sampling is a viable option for potentiometric sensors to be used for the determination of analytes in samples with high solid-to-liquid ratios. Unfortunately, heavy metal sensitive electrodes cannot be easily integrated with paper-based solution sampling as heavy metals have strong physicochemical adsorption affinity towards paper substrates. In this work, paper substrates were modified with an ion-selective membrane (ISM) cocktail (used for the preparation of Pb2+-ion-selective electrodes (ISEs)) and coupled with model heavy metal Pb2+-ISEs. It was found that the super-Nernstian response of Pb2+-ISEs was eliminated when 10 to 50 mg ml-1 of the ISM cocktail was used for the modification of paper substrates. The modification of the paper substrates by Pb2+-ISM allowed the elimination of adsorption sites. In addition, it resulted in an improvement of sensor performance in terms of their detection limits to be similar to those for conditioned electrodes in standard beaker-based measurements. It is believed that the elimination of super-Nernstian response of the electrodes and improving the potentiometric responses and detection limits of ISEs were attributed to the compatibility improvement of the paper substrates and Pb2+-ISEs to the same type of ISM.

CryoWriter: a blotting free cryo-EM preparation system with a climate jet and cover-slip injector.

Electron microscopy (EM) introduced a fast and lasting change to structural and cellular biology. However, the sample preparation is still the bottleneck in the cryogenic electron microscopy (cryo-EM) workflow. Classical specimen preparation methods employ a harsh paper-blotting step, and the protein particles are exposed to a damaging air-water interface. Therefore, improved preparation strategies are urgently needed. Here, we present an amended microfluidic sample preparation method, which entirely avoids paper blotting and allows the passivation of the air-water interface during the preparation process. First, a climate jet excludes oxygen from the sample environment and controls the preparation temperature by varying the relative humidity of the grid environment. Second, the integrated "coverslip injector" allows the modulation of the air-water interface of the thin sample layer with effector molecules. We will briefly discuss the climate jet's effect on the stability and dynamics of the sample thin films. Furthermore, we will address the coverslip injector and demonstrate significant improvement in the sample quality.

Fast dopamine detection based on evanescent wave detection platform

A compact evanescent wave detection platform (EWDP) is developed for the detection of fluorescence gold nanoclusters. The EWDP employs a simple optical system and a Si-based photodetector SOP-1000 assembly to improve the optical efficiency and detection sensitivity. A microfluidic sample cell is also used to decrease the amount of analyte to 200 μL (The volume of sample cell is really about 30 μL). On this basis, we design a strategy for detecting dopamine (DA) based on the photoinduced electron transfer (PET) quenching mechanism. By introduction of tyrosinase (TYR) during the detection, the testing time is shortened to 1 min. The fluorescence emission signal decreased dramatically and the quenching ratio (F0–F)/F0 is linearly related to the concentration of DA in the range of 0.03–60 μM with a detection limit of 0.03 μM. Additionally, this detection platform has potential applications for DA fast detection in the microsamples.

Extremely Sensitive Microwave Microfluidic Dielectric Sensor Using a Transmission Line Loaded with Shunt LC Resonators

In this paper, a very high sensitivity microwave-based planar microfluidic sensor is presented. Sensitivity enhancement is achieved and described theoretically and experimentally by eliminating any extra parasitic capacitance not contributing to the sensing mechanism. The sensor consists of a microstrip transmission line loaded with a series connected shunt LC resonator. A microfluidic channel is attached to the area of the highest electric field concentration. The electric field distribution and, therefore, the resonance characteristics are modified by applying microfluidic dielectric samples to the sensing area. The sensor performance and working principle are described through a circuit model analysis. A device prototype is fabricated, and experimental measurements using water/ethanol and water/methanol solutions are presented for validation of the sensing mathematical model.

Multistage Digital-to-Analogue Chip Based on a Weighted Flow Resistance Network for Soft Actuators.

A control chip with a multistage flow-rate regulation function based on the correlation between the flow resistance and flow rate has been developed in this article. Compared with the traditional proportional solenoid valve, this kind of flow valve based on microfluidic technology has the characteristics of being light-weight and having no electric drive. It solves such technical problems as how the current digital microfluidic chip can only adjust the flow switch, and the adjustment of the flow rate is difficult. To linearize the output signal, we propose a design method of weighted resistance. The output flow is controlled by a 4-bit binary pressure signal. According to the binary value of the 4-bit pressure signal at the input, the output can achieve 16-stage flow adjustment. Furthermore, we integrate the three-dimensional flow resistance network, multilayer structure microvalve, and parallel fluid network into a single chip by using 3D printing to obtain a modular flow control unit. This structure enables the microflow control signal to be converted from a digital signal to an analogue signal (DA conversion), and is suitable for microflow driving components, such as in microfluidic chip sampling systems and proportional mixing systems. In the future, we expect this device to even be used in the automatic control system of a miniature pneumatic soft actuator.