ABSTRACT
A matrix in highly complex samples can cause adverse effects on the trace analysis of targeted organic compounds. A suitable separation of the target analyte(s) and matrix before the instrumental analysis is often a vital step for which chromatographic cleanup methods remain one of the most frequently used strategies, particularly high-performance liquid chromatography (HPLC). The lack of a simple real-time detection technique that can quantify the entirety of the matrix during this step, especially with gradient solvents, renders optimization of the cleanup challenging. This paper, along with a companion one, explores the possibilities and limitations of quartz crystal microbalance (QCM) dry-mass sensing for quantifying complex organic matrices during gradient HPLC. To this end, this work coupled a QCM and a microfluidic spray dryer with a commercial HPLC system using a flow splitter and developed a calibration and data processing strategy. The system was characterized in terms of detection and quantification limits, with LOD = 4.3-15 mg/L and LOQ = 16-52 mg/L, respectively, for different eluent compositions. Validation of natural organic matter in an environmental sample against offline total organic carbon analysis confirmed the approach's feasibility, with an absolute recovery of 103 ± 10%. Our findings suggest that QCM dry-mass sensing could serve as a valuable tool for analysts routinely employing HPLC cleanup methods, offering potential benefits across various analytical fields.
ABSTRACT
For the treatment of human immunodeficiency virus (HIV)-infected patients, the regular assessment of the immune status is indispensable. The quantification of CD4+ T lymphocytes in blood by gold standard optical flow cytometry is not point-of-care testing (POCT) compatible. This incompatibility is due to unavoidable pre-analytics, expensive and bulky optics with limited portability, and complex workflow integration. Here, we propose a non-optical, magnetic flow cytometry (MFC) workflow that offers effortless integration opportunities, including minimal user interaction, integrated sample preparation and up-concentration, and miniaturization. Furthermore, we demonstrate immunomagnetic CD4+ T lymphocyte labeling in whole blood with subsequent quantification using sheath-less MFC. Showing linearity over two log scales and being largely unimpaired by hematocrit, evidence is provided for POCT capabilities of HIV patients.
ABSTRACT
Optical flow cytometry is used as the gold standard in single cell function diagnostics with the drawback of involving high complexity and operator costs. Magnetic flow cytometers try to overcome this problem by replacing optical labeling with magnetic nanoparticles to assign each cell a magnetic fingerprint. This allows operators to obtain rich cell information from a biological sample with minimal sample preparation at near in-vivo conditions in a decentralized environment. A central task in flow cytometry is the determination of cell concentrations and cell parameters, e.g. hydrodynamic diameter. For the acquisition of this information, signal processing is an essential component. Previous approaches mainly focus on the processing of one-cell signals, leaving out superimposed signals originating from cells passing the magnetic sensors in close proximity. In this work, we present a framework for joint cell/particle detection and analysis, which is capable of processing one-cell as well as multi-cell signals. We employ deep learning and compressive sensing in this approach, which involves the minimization of an adaptive norm. We evaluate our method on simulated and experimental signals, the latter being obtained with polymer microparticles. Our results show that the framework is capable of counting cells with a relative error smaller than 2%. Inference of cell parameters works reliably at both low and high noise levels.
Subject(s)
Data Compression , Signal Processing, Computer-Assisted , Flow Cytometry/methodsABSTRACT
Time-of-flight (TOF) magnetic sensing of rolling immunomagnetically-labeled cells offers great potential for single cell function analysis at the bedside in even optically opaque media, such as whole blood. However, due to the spatial resolution of the sensor and the low flow rate regime required to observe the behavior of rolling cells, the concentration range of such a workflow is limited. Potential clinical applications, such as testing of leukocyte function, require a cytometer which can cover a cell concentration range of several orders of magnitude. This is a challenging task for an integrated dilution-free workflow, as for high cell concentrations coincidences need to be avoided, while for low cell concentrations sufficient statistics should be provided in a reasonable time-to-result. Here, we extend the spatial bandwidth of a magnetoresistive sensor with an adaptive and integratable workflow concept combining mechanical and magnetophoretic guiding of magnetically labeled targets for in-situ enrichment over a dynamic concentration range of 3 orders of magnitude. We achieve hybrid integration of the enrichment strategy in a cartridge mold and a giant-magnetoresistance (GMR) sensor in a functionalized Quad Flat No-Lead (QFN) package, which allows for miniaturization of the Si footprint for potential low-cost bedside testing. The enrichment results demonstrate that TOF magnetic flow cytometry with adaptive particle focusing can match the clinical requirements for a point-of-care (POC) cytometer and can potentially be of interest for other sheath-less methodologies requiring workflow integration.
Subject(s)
Biosensing Techniques , Flow Cytometry/methods , Microfluidic Analytical Techniques , Point-of-Care Systems , Single-Cell Analysis/methodsABSTRACT
Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis.
Subject(s)
Erythrocytes/cytology , Flow Cytometry/instrumentation , Flow Cytometry/methods , Magnetics , Microfluidic Analytical Techniques/instrumentation , Humans , HydrodynamicsABSTRACT
Rapid and specific rare cell detection for point-of-care testing requires an integration of the sample preparation for flow cytometry. To achieve such a challenging goal we have developed a magnetic flow cytometry technique which applies magnetophoresis to perform cell enrichment, focusing, and background elimination in a single step. Time-of-flight measurements are performed with integrated magnetic sensors to detect specifically cancer cells and cell diameters in whole blood.