Sample Delivery System Research Articles

Developing an in situ LED irradiation system for small-angle X-ray scattering at B21, Diamond Light Source.

Beamline B21 at the Diamond Light Source synchrotron in the UK is a small-angle X-ray scattering (SAXS) beamline that specializes in high-throughput measurements via automated sample delivery systems. A system has been developed whereby a sample can be illuminated by a focused beam of light coincident with the X-ray beam. The system is compatible with the highly automated sample delivery system at the beamline and allows a beamline user to select a light source from a broad range of wavelengths across the UV and visible spectrum and to control the timing and duration of the light pulse with respect to the X-ray exposure of the SAXS measurement. The intensity of the light source has been characterized across the wavelength range enabling experiments where a quantitative measure of dose is important. Finally, the utility of the system is demonstrated via measurement of several light-responsive samples.

Systematic Research and Application of a 5G Medical Unmanned Aerial Vehicle to Deliver COVID-19 Nucleic Acid Samples.

The purpose of this study was to investigate the feasibility and effectiveness of COVID-19 throat swab samples delivered by medical drones in epidemic prevention and control. This study was carried out in both southern and northern hospital districts of the Affiliated Hospital of Jiangnan University from May to October 2022. The main participants were the Affiliated Hospital of Jiangnan University and Zhejiang Antwork Technology Co., Ltd. We first constructed an urban medical unmanned aerial vehicle (UAV) delivery system and developed a UAV-specific storage box for COVID-19 samples. The UAV system was used to transport COVID-19 throat swab samples from the northern hospital district to the southern hospital district, and the following indexes were obtained: (1) flight time of COVID-19 samples delivered by UAV, (2) real-time temperature of COVID-19 nucleic acid samples during transportation, and (3) the time of distribution of COVID-19 nucleic acid samples by road traffic as measured using the Baidu Maps application, compared with the flight time of UAV. The COVID-19 sample delivery system for urban medical UAV mainly consists of intelligent logistics UAV, low-temperature COVID-19 throat swab sample storage box, unmanned logistics hub, and cloud operation control platform. The flight distance between the northern and southern districts of the Affiliated Hospital of Jiangnan University was 10 km, and the ground distance was 24 km. From May 11 to October 28, 2022, a total of 1,190 UAV flights occurred. The average flight time was 13 minutes, which was 40 to 70 minutes faster than the average road travel time required for manual delivery of COVID-19 throat swab samples. At different time points in the day, UAV delivery efficiency increased by 67.5% to 82%. The use of 5G with the Internet of Things and UAV technology to deliver nucleic acid samples has the characteristics of fast speed, being unaffected by ground traffic conditions, and the ability to ensure the safety of nucleic acid samples in the transportation process, which is worthy of further study.

X-ray spectroscopy station for sample characterization at ELI Beamlines

X-ray spectroscopy is a demanded tool across multiple user communities. Here we report on a new station for X-ray emission spectroscopy at the Extreme Light Infrastructure Beamlines Facility. The instrument utilizes the von Hamos geometry and works with a number of different sample types, notably including liquid systems. We demonstrate a simple and reliable method for source position control using two cameras. This approach addresses energy calibration dependence on sample position, which is a characteristic source of measurement uncertainty for wavelength dispersive spectrometers in XES arrangement. We also present a straightforward procedure for energy calibration of liquid and powder samples to a thin film reference. The developed instrumentation enabled us to perform the first experimental determination of the Kα lines of liquidized K3Fe(CN)6 as well as powdered and liquidized FeNH4(SO4)2. Finally, we report on proof-of-principle use of a colliding jet liquid sample delivery system in an XES experiment.

A numerical study of gas focused non-Newtonian micro-jets

The present numerical study assesses the jet length, diameter and velocity of various non-Newtonian power-law fluids of a gas dynamic virtual nozzle. A related two-phase flow problem is formulated within the mixture framework and solved with the finite volume method and volume of fluid interface treatment in axisymmetry. The process parametric range allows the incompressible laminar flow assumption. A comprehensive jet characteristics analysis is carried out in a typical micro-nozzle configuration for a range of shear-thinning to shear-thickening fluids with power law indices 0.5 ≤ n < 1.5, gas mass flow rate of 10 mg/min and liquid volumetric flow rate of 43 µl/min, resulting in a gas Reynolds number of 130 with Reynolds and Weber numbers for a reference water jet being 90 and 10, respectively. It is observed that jets from shear-thinning fluids (0.5 ≤ n < 1.0) tend to be thicker, longer, and slower when compared with the shear-thickening fluids (1.0 < n ≤ 1.5). A dripping-jetting phase diagram of the nozzle is constructed by varying the power law index, gas and liquid flow rates in the range 0.9–1.1, 5–15 mg/min and 5–50 µl/min, respectively. It is observed that the area of stable jetting decreases with the increase of the power law index. The obtained novel information on the behaviour of non-Newtonian gas-focused micro-jets provides a possible new dimension for tailoring the serial crystallography sample delivery systems where the micro-jets carry dispersed crystals into an X-ray beam.

Development of spinning-disk solid sample delivery system for high-repetition rate x-ray free electron laser experiments.

X-ray free-electron lasers (XFELs) deliver intense x-ray pulses that destroy the sample in a single shot by a Coulomb explosion. Experiments using XFEL pulse trains or the new generation of high-repetition rate XFELs require rapid sample replacement beyond those provided by the systems now used at low repletion-rate XFELs. We describe the development and characterization of a system based on a spinning disk to continuously deliver a solid sample into an XFEL interaction point at very high speeds. We tested our system at the Linac Coherent Light Source and European XFEL hard x-ray nano-focus instruments, employing it to deliver a 25 μm copper foil sample, which can be used as a gain medium for stimulated x-ray emission for the proposed x-ray laser oscillator.

(Invited) Translational Applications of Nanostructured Biosensors: Diagnostics at the Point of Care

Development of diagnostic devices with clinically relevant sensitivity and rapidity is highly desirable for decreasing the delay between diagnosis and treatment. Diagnostic inefficiency permeates multiple medical fields including infectious diseases and antimicrobial resistance (both recognized by WHO among paramount threats and research priorities). Molecular detection is also central to cancer, where therapies are often out of step with disease complexity and progression. The respective challenges may be addressed through the application of nanomaterial and high-throughput devices that offer unique advantages. In Mahshid Lab, we develop novel paradigms in point of care diagnosis via synergistically combining innovative nanostructured sensors with fluidic sample delivery systems and biomolecular assay capabilities (Nano/Bio diagnostic devices).From an engineering perspective, the lab seeks to use the remarkable intrinsic properties of novel nanomaterials, to render them capable of sensing the specific biomolecules. Such miniaturized sensors could be integrated with automated lab-chip devices and deployed to diagnose molecular changes in biological systems and in disease such as cancer (by targeting new cancer biomarkers) or to detect infectious agents in biological samples, e.g. in blood, saliva and urine. From a health industry perspective, we target the advancement of the automated and portable tools for in-field testing, remote locations and hospitals in close collaboration with clinicians to validate the devices with clinical samples. In particular and in the context of infectious disease, Mahshid lab has developed SALIVERA analogous to a qPCR, and NFluidEX analogous to a glucometer, that enabled rapid portable automated monitoring of SARS-CoV-2 infection in patient saliva and antibodies in patient blood, respectively. In the context of cancer, Mahshid lab has developed MoSERS, anon-chip approach for molecular profiling of extracellular vesicles (a new cancer biomarker)on-chip approach for molecular profiling of extracellular vesicles (a new cancer biomarker)in plasma and cerebrospinal fluid of glioblastoma patients. The proposed hybrid devices are capable of working with small sample volumes and precise dosing of reagents, enabling the transition to a portable diagnostic tool.

Tape drive sample delivery system for time-resolved serial femtosecond crystallography

Tape drive sample delivery system for time-resolved serial femtosecond crystallography

Hard X-ray emission spectroscopy in liquids using MHz XFEL source and JUNGFRAU detectors

X-ray free-electron lasers (XFEL) provide unique opportunities for hard X-ray emission spectroscopy (XES) in solution phase by enabling femtosecond temporal resolution. The high brilliance and MHz repetition rate of such sources drive the development of specialized sample delivery systems to overcome the challenges of sample degradation, as well as major advances of multiple-gain detectors to fully utilize the high dynamic range required for X-ray detection. This paper addresses the experimental limitations that arise in a typical XES experiment performed on fast liquid jets at the MHz repetition rate of the European XFEL. Using a low-noise gain-switching JUNGFRAU detector allows taking advantage of the high dynamic range and high sensitivity, but restricts a single-shot detection due to the limited frame rate. The gain transition regions of the detector response may exhibit nonlinear behavior that can be identified and efficiently corrected using the internal reference-based normalization and linearization approaches described here.

Sample Delivery Systems for Serial Femtosecond Crystallography at the PAL-XFEL

Serial femtosecond crystallography (SFX) using an X-ray free electron laser (XFEL) enables the determination of room-temperature structures without causing radiation damage. Using an optical pump-probe or mix-and-injection, SFX enables the intermediate state visualization of a molecular reaction. In SFX experiments, serial and stable microcrystal delivery to the X-ray interaction point is vital for reasonable data collection and efficient beam time. The Pohang Accelerator Laboratory X-ray Free Electron Laser (PAL-XFEL) facility established SFX instruments at a nanocrystallography and coherent imaging (NCI) experimental station. Various sample delivery methods, including injection, fixed-target scanning, and hybrid methods, have been developed and applied to collect XFEL diffraction data. Herein, we report the currently available sample delivery methods for SFX at the NCI experimental station at the PAL-XFEL. This article will help PAL-XFEL users access the SFX system for their experiments.

A small-angle X-ray scattering study of red blood cells in continuous flow.

Owing to their large penetration depth and high resolution, X-rays are ideally suited to study structures and structural changes within intact biological cells. For this reason, X-ray-based techniques have been used to investigate adhesive cells on solid supports. However, these techniques cannot easily be transferred to the investigation of suspended cells in flow. Here, an X-ray compatible microfluidic device that serves as a sample delivery system and measurement environment for such studies is presented. As a proof of concept, the microfluidic device is applied to investigate chemically fixed bovine red blood cells by small-angle X-ray scattering (SAXS). A very good agreement is found between in-flow and static SAXS data. Moreover, the data are fitted with a hard-sphere model and screened Coulomb interactions to obtain the radius of the protein hemoglobin within the cells. Thus, the utility of this device for studying suspended cells with SAXS in continuous flow is demonstrated.

Coherent diffractive imaging of proteins and viral capsids: simulating MS SPIDOC

MS SPIDOC is a novel sample delivery system designed for single (isolated) particle imaging at X-ray Free-Electron Lasers that is adaptable towards most large-scale facility beamlines. Biological samples can range from small proteins to MDa particles. Following nano-electrospray ionization, ionic samples can be m/z-filtered and structurally separated before being oriented at the interaction zone. Here, we present the simulation package developed alongside this prototype. The first part describes how the front-to-end ion trajectory simulations have been conducted. Highlighted is a quadrant lens; a simple but efficient device that steers the ion beam within the vicinity of the strong DC orientation field in the interaction zone to ensure spatial overlap with the X-rays. The second part focuses on protein orientation and discusses its potential with respect to diffractive imaging methods. Last, coherent diffractive imaging of prototypical T = 1 and T = 3 norovirus capsids is shown. We use realistic experimental parameters from the SPB/SFX instrument at the European XFEL to demonstrate that low-resolution diffractive imaging data (q < 0.3 nm−1) can be collected with only a few X-ray pulses. Such low-resolution data are sufficient to distinguish between both symmetries of the capsids, allowing to probe low abundant species in a beam if MS SPIDOC is used as sample delivery.

Increased hemolysis rate in plasma tubes after implementation of a fully automated sample delivery and acceptance system

Abstract Objectives Automated sample delivery and laboratory acceptance systems (PTAS) may influence the hemolysis rate of blood samples due to g-forces, abrupt acceleration, and rapid deceleration. However, quantitative data regarding the rate of hemolysis in PTAS is limited. To fill this void, the effect of a pneumatic tube in combination with an acceptance system (PTAS) on the hemolysis rate was investigated in this study. Methods Lithium heparin plasma tubes were transported from different clinical departments to the hospital’s laboratory (a) by employees or (b) with an automated PTAS and analyzed for the presence of hemolysis based on a hemolysis index (HI) of &gt;25. Hemolysis indices of 68.513 samples were retrieved from the laboratory information system before and after installation of the PTAS and were subjected to statistical analysis. Results A total of 32.614 samples were transported by employees, of which 3.815 samples (11.70%) were hemolytic, and 9.441 out of 35.899 samples delivered by PTAS (26.30%) were hemolytic. After the implementation of the PTAS, hemolysis rates increased in all departments. Conclusions Automated PTAS are associated with increased hemolysis rates. This has implications for routine patient management and should be considered for the transportation of samples used for the determination of hemolysis-sensitive laboratory parameters.

Rapid and efficient room-temperature serial synchrotron crystallography using the CFEL TapeDrive.

Serial crystallography at conventional synchrotron light sources (SSX) offers the possibility to routinely collect data at room temperature using micrometre-sized crystals of biological macromolecules. However, SSX data collection is not yet as routine and currently takes significantly longer than the standard rotation series cryo-crystallography. Thus, its use for high-throughput approaches, such as fragment-based drug screening, where the possibility to measure at physio-logical temperatures would be a great benefit, is impaired. On the way to high-throughput SSX using a conveyor belt based sample delivery system - the CFEL TapeDrive - with three different proteins of biological relevance (Klebsiella pneumoniae CTX-M-14 β-lactamase, Nectria haematococca xylanase GH11 and Aspergillus flavus urate oxidase), it is shown here that complete datasets can be collected in less than a minute and only minimal amounts of sample are required.

Upgraded Combined Inject-and-Transfer System for Serial Femtosecond Crystallography

Serial femtosecond crystallography (SFX) using an X-ray free-electron laser (XFEL) can be applied to determine the room-temperature structure of target molecules while minimizing radiation damage and visualizing molecular dynamics. In SFX, a sample delivery system is required to deliver microcrystals to the XFEL beam path in a serial manner. We recently developed a sample delivery method, the combined inject-and-transfer system (BITS), which is a hybrid method based on the injector and fixed-target scanning approach. In this study, we introduced recently upgraded hardware to move the injection needle in the direction of the XYZ-axis and a graphic user interface for user motion control. Furthermore, we report that the viscous solution containing 10% (w/v) PEG 3350 or PEG 6000 that is widely used for protein crystallization can be stably deposited on polyimide film with a hydrophobic surface without any special treatment. Moreover, the development of an inject-and-diffuse method for time-resolved studies with liquid applications in the BITS and its preliminary results are reported. This study provides up-to-date instrument information to SFX users using BITS and provides insights to instrument developers for SFX.

Acoustic Focusing of Protein Crystals for In-Line Monitoring and Up-Concentration during Serial Crystallography

Serial femtosecond crystallography (SFX) has become one of the standard techniques at X-ray free-electron lasers (XFELs) to obtain high-resolution structural information from microcrystals of proteins. Nevertheless, reliable sample delivery is still often limiting data collection, as microcrystals can clog both field- and flow-focusing nozzles despite in-line filters. In this study, we developed acoustic 2D focusing of protein microcrystals in capillaries that enables real-time online characterization of crystal size and shape in the sample delivery line after the in-line filter. We used a piezoelectric actuator to create a standing wave perpendicular to the crystal flow, which focused lysozyme microcrystals into a single line inside a silica capillary so that they can be imaged using a high-speed camera. We characterized the acoustic contrast factor, focus size, and the coaxial flow lines and developed a splitting union that enables up-concentration to at least a factor of five. The focus size, flow rates, and geometry may enable an upper limit of up-concentration as high as 200 fold. The novel feedback and concentration control could be implemented for serial crystallography at synchrotrons with minor modifications. It will also aid the development of improved sample delivery systems that will increase SFX data collection rates at XFELs, with potential applications to many proteins that can only be purified and crystallized in small amounts.

Radiation Damage of Polydimethylsiloxane and Polyimide by X-ray Free-Electron Laser

A crystal delivery system is essential in serial femtosecond crystallography experiments using an X-ray free-electron laser (XFEL). Investigating the XFEL-induced radiation damage to materials potentially applicable to sample delivery devices is vital for developing a sample delivery system. In this study, we investigated the radiation damage caused by an XFEL to polydimethylsiloxane (PDMS) and polyimide (PI), which are widely used as sample delivery materials in synchrotron X-rays. Upon XFEL exposure, the PDMS film produced irregularly shaped and sized holes, whereas the PI film produced relatively regular shaped and sized holes. When XFELs were exposed to the channel of the PDMS-based microfluidic device, holes were generated on the film by the radiation damage and the microfluidic device and the internal channel region were structurally destroyed. The PI-based microfluidic device experienced no structural destruction, except for the holes generated by the XFEL. However, as the XFELs were continuously exposed, bubbles generated from the solution due to radiation damage; the accumulation of these bubbles interfered with the path of the inner channel of the microfluidic device. Our results will not only help understand the phenomenon of radiation damage of PDMS and PI films by XFEL, but also provide insight into the directions to pursue in developing applications of PDMS and PI films in XFEL studies.

Towards real-time analysis of liquid jet alignment in serial femtosecond crystallography.

Liquid sample delivery systems are used extensively for serial femtosecond crystallography at X-ray free-electron lasers (XFELs). However, misalignment of the liquid jet and the XFEL beam leads to the X-rays either partially or completely missing the sample, resulting in sample wastage and a loss of experiment time. Implemented here is an algorithm to analyse optical images using machine vision to determine whether there is overlap of the X-ray beam and liquid jet. The long-term goal is to use the output from this algorithm to implement an automated feedback mechanism to maintain constant alignment of the X-ray beam and liquid jet. The key elements of this jet alignment algorithm are discussed and its performance is characterized by comparing the results with a manual analysis of the optical image data. The success rate of the algorithm for correctly identifying hits is quantified via a similarity metric, the Dice coefficient. In total four different nozzle designs were used in this study, yielding an overall Dice coefficient of 0.98.

Predicting the oxidation states of Mn ions in the oxygen-evolving complex of photosystem II using supervised and unsupervised machine learning

Serial Femtosecond Crystallography at the X-ray Free Electron Laser (XFEL) sources enabled the imaging of the catalytic intermediates of the oxygen evolution reaction of Photosystem II (PSII). However, due to the incoherent transition of the S-states, the resolved structures are a convolution from different catalytic states. Here, we train Decision Tree Classifier and K-means clustering models on Mn compounds obtained from the Cambridge Crystallographic Database to predict the S-state of the X-ray, XFEL, and CryoEM structures by predicting the Mn’s oxidation states in the oxygen-evolving complex. The model agrees mostly with the XFEL structures in the dark S1 state. However, significant discrepancies are observed for the excited XFEL states (S2, S3, and S0) and the dark states of the X-ray and CryoEM structures. Furthermore, there is a mismatch between the predicted S-states within the two monomers of the same dimer, mainly in the excited states. We validated our model against other metalloenzymes, the valence bond model and the Mn spin densities calculated using density functional theory for two of the mismatched predictions of PSII. The model suggests designing a more optimized sample delivery and illumiation systems are crucial to precisely resolve the geometry of the advanced S-states to overcome the noncoherent S-state transition. In addition, significant radiation damage is observed in X-ray and CryoEM structures, particularly at the dangler Mn center (Mn4). Our model represents a valuable tool for investigating the electronic structure of the catalytic metal cluster of PSII to understand the water splitting mechanism.

Review about Powerful Combinations of Advanced and Hyphenated Sample Introduction Techniques with Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) for Elucidating Trace Element Species in Pathologic Conditions on a Molecular Level.

Element analysis in clinical or biological samples is important due to the essential role in clinical diagnostics, drug development, and drug-effect monitoring. Particularly, the specific forms of element binding, actual redox state, or their spatial distribution in tissue or in single cells are of interest in medical research. This review summarized exciting combinations of sophisticated sample delivery systems hyphenated to inductively coupled plasma-mass spectrometry (ICP-MS), enabling a broadening of information beyond the well-established outstanding detection capability. Deeper insights into pathological disease processes or intracellular distribution of active substances were provided, enabling a better understanding of biological processes and their dynamics. Examples were presented from spatial elemental mapping in tissue, cells, or spheroids, also considering elemental tagging. The use of natural or artificial tags for drug monitoring was shown. In the context of oxidative stress and ferroptosis iron, redox speciation gained importance. Quantification methods for Fe2+, Fe3+, and ferritin-bound iron were introduced. In Wilson’s disease, free and exchangeable copper play decisive roles; the respective paragraph provided information about hyphenated Cu speciation techniques, which provide their fast and reliable quantification. Finally, single cell ICP-MS provides highly valuable information on cell-to-cell variance, insights into uptake of metal-containing drugs, and their accumulation and release on the single-cell level.

An advanced automation platform coupled with mass spectrometry for investigating in vitro human skin permeation of UV filters and excipients in sunscreen products.

Systemic absorption of UV-filtering chemicals following topical application of sunscreens may present a safety concern. The Food and Drug Administration (FDA) had recommended an in vitro skin permeation test (IVPT) to evaluate the potential of this safety risk for the evaluation of sunscreens prior to clinical studies. Therefore, a sensitive and robust bioanalytical method(s) were required for IVPT studies of different topical sunscreen products. An analytical procedure to quantitate sunscreen UV-filtering components and excipients in IVPT samples including avobenzone, octocrylene, oxybenzone, ecamsule, methylparaben and propylparaben was developed employing a RapidFire 360 robotic sample delivery system coupled with a triple quadrupole mass spectrometer. The analytical procedure was developed and validated according to the requirements of the FDA Bioanalytical Method Validation Guidance for Industry (2018). The analytical method provided a turnaround time of 12 seconds per sample and was determined to be accurate, precise, specific, and linear over the corresponding analytical ranges. The validated method was successfully applied for two IVPT studies for evaluating the skin permeation potential of UV-filtering chemicals and assisting with the selection of the sunscreen products for the clinical study conducted by the FDA. This work highlights the first analytical procedure that has applied a non-chromatographic-MS/MS automation platform to an in vitro biopharmaceutics study. The analytical platform simultaneously quantitated four UV filters and two excipients in complex media to evaluate their permeation in IVPT studies. The sample throughput and analytical performance of advanced automation platforms indicate their analytical procedure has the potential to significantly advance the efficiency of IVPT studies to evaluate permeation of a wide variety of UV chemical filters and excipients for topical OTC sunscreen products.