Continuous Perfusion Experiments on 3D Cell Proliferation in Acoustic Levitation.

An acoustofluidic trap is used for accurate 3D cell proliferation and cell function analysis in levitation. The prototype trap can be integrated with any microscope setup, allowing continuous perfusion experiments with temperature and flow control under optical inspection. To describe the trap function, we present a mathematical and FEM-based COMSOL model for the acoustic mode that defines the nodal position of trapped objects in the spherical cavity aligned with the microscope field of view and depth of field. Continuous perfusion experiments were conducted in sterile conditions over 55 h with a K562 cell line, allowing for deterministic monitoring. The acoustofluidic platform allows for rational in vitro cell testing imitating in vivo conditions such as cell function tests or cell-cell interactions.

Towards a Point-of-Care Test of CD4+ T Lymphocyte Concentrations for Immune Status Monitoring with Magnetic Flow Cytometry.

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.

Grease the gears: how lubrication of syringe pumps impacts microfluidic flow precision.

Pumps are indispensable for analytical applications and ensure controlled fluid movement. Syringe pumps are among today's most prevalent liquid delivery systems, especially for high-pressure, stable, low-flow-rate microfluidic applications. Due to moving mechanical parts of the assembly, regular maintenance is essential to ensure reliable operation and flow rates. However, lubrication of the mechanics is easily overlooked because the research focuses on novel analytical applications rather than on the maintenance of pumps. Here, we investigate the lubrication of the syringe pump guide rods with its effect on the flow rate stability after regular pump cleaning from contaminations. The guide rods of syringe pumps were thoroughly cleaned from any lubricant, and the flow rate for specified flowrates between 5 and 100 µL min-1 was measured, revealing tremendous flow rate fluctuations with a coefficient of variation (CV) value up to 0.34. In contrast, flow rate measurements of syringe pumps with lubricated guide rods show a five-fold smoother flow rate fluctuation depending on the specified flow rate with CV values below 0.07. These differences in flow rate's CV were most significant for pressure drops below 500 mbar, relevant for many lab-on-a-chip applications. In summary, we emphasize the awareness of lubricating moving parts of syringe pumps to achieve constant flow rates, minimize wear, and ensure the reliable operation of, for instance, accurate lab-on-a-chip workflows.

Platelet aggregates detected using quantitative phase imaging associate with COVID-19 severity.

BACKGROUND: The clinical spectrum of acute SARS-CoV-2 infection ranges from an asymptomatic to life-threatening disease. Considering the broad spectrum of severity, reliable biomarkers are required for early risk stratification and prediction of clinical outcomes. Despite numerous efforts, no COVID-19-specific biomarker has been established to guide further diagnostic or even therapeutic approaches, most likely due to insufficient validation, methodical complexity, or economic factors. COVID-19-associated coagulopathy is a hallmark of the disease and is mainly attributed to dysregulated immunothrombosis. This process describes an intricate interplay of platelets, innate immune cells, the coagulation cascade, and the vascular endothelium leading to both micro- and macrothrombotic complications. In this context, increased levels of immunothrombotic components, including platelet and platelet-leukocyte aggregates, have been described and linked to COVID-19 severity. METHODS: Here, we describe a label-free quantitative phase imaging approach, allowing the identification of cell-aggregates and their components at single-cell resolution within 30 min, which prospectively qualifies the method as point-of-care (POC) testing. RESULTS: We find a significant association between the severity of COVID-19 and the amount of platelet and platelet-leukocyte aggregates. Additionally, we observe a linkage between severity, aggregate composition, and size distribution of platelets in aggregates. CONCLUSIONS: This study presents a POC-compatible method for rapid quantitative analysis of blood cell aggregates in patients with COVID-19.

The human body produces a series of immune responses when it gets infected with SARS-CoV-2, the virus that causes COVID-19. One of these responses involves platelets, the blood clotting factor sticking to immune cells to form cell aggregates in the bloodstream. We aimed to understand the significance of these cell aggregates in COVID-19 disease progression. A quantitative imaging approach was used to investigate the number and components of these cell aggregates in SARS-CoV-2 infected patient blood. We observed blood from severe COVID-19 patients was associated with higher numbers and specific composition of cell aggregates. Our method can potentially support the risk stratification of severe patients to prevent complications in COVID-19 and other medical disorders, where immune cells are shown to aggregate.

Fully Automated Chemiluminescence Microarray Analysis Platform for Rapid and Multiplexed SARS-CoV-2 Serodiagnostics.

Lateral-flow immunoassays and laboratory diagnostic tests like enzyme-linked immunosorbent assays (ELISAs) are powerful diagnostic tools to help fight the COVID-19 pandemic using them as antigen or antibody tests. However, the need emerges for alternative bioanalytical systems that combine their favorable features─simple, rapid, and cost-efficient point-of-care (POC) analysis of lateral-flow immunoassays and higher reliability of laboratory tests─while eliminating their disadvantages (limited sensitivity and specificity of lateral-flow assays and prolonged time and work expenditure of laboratory analysis). An additional need met by only a few tests is multiplexing, allowing for the analysis of several immunorecognition patterns at the same time. We herein present a strategy to combine all desirable attributes of the different test types by means of a flow-based chemiluminescence microarray immunoassay. Laminated polycarbonate microarray chips were developed for easy production and subsequent application in the fully automated microarray analysis platform MCR-R, where a novel flow cell design minimizes the sample volume to 40 µL. This system was capable of detecting IgG antibodies to SARS-CoV-2 with 100% sensitivity and specificity using recombinant antigens for the SARS-CoV-2 spike S1 protein, nucleocapsid protein, and receptor binding domain. The analysis was accomplished within under 4 min from serum, plasma, and whole blood, making it also useful in POC settings. Additionally, we showed the possibility of serosurveillance after infection or vaccination to monitor formerly unnoticed breakthrough infections in the population as well as to detect the need for booster vaccination after the natural decline of the antibody titer below detectable levels. This will help in answering pressing questions on the importance of the antibody response to SARS-CoV-2 that so far remain open. Additionally, even the sequential detection of IgM and IgG antibodies was possible, allowing for statements on the time response of an infection. While our serodiagnostic application focuses on SARS-CoV-2, the same approach is easily adjusted to other diseases, making it a powerful tool for future serological testing.

Joint Particle Detection and Analysis by a CNN and Adaptive Norm Minimization Approach.

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.

Capacitive Sensor and Alternating Drive Mixing for Microfluidic Applications Using Micro Diaphragm Pumps.

Microfluidic systems are of paramount importance in various fields such as medicine, biology, and pharmacy. Despite the plethora of methods, accurate dosing and mixing of small doses of liquid reagents remain challenges for microfluidics. In this paper, we present a microfluidic device that uses two micro pumps and an alternating drive pattern to fill a microchannel. With a capacitive sensor system, we monitored the fluid process and controlled the micro pumps. In a first experiment, the system was set up to generate a 1:1 mixture between two fluids while using a range of fluid packet sizes from 0.25 to 2 µL and pumping frequencies from 50 to 100 Hz. In this parameter range, a dosing accuracy of 50.3 ± 0.9% was reached, validated by a gravimetric measurement. Other biased mixing ratios were tested as well and showed a deviation of 0.3 ± 0.3% from the targeted mixing ratio. In a second experiment, Trypan blue was used to study the mixing behavior of the system. Within one to two dosed packet sets, the two reagents were reliably mixed. The results are encouraging for future use of micro pumps and capacitive sensing in demanding microfluidic applications.

Microfluidic Cell Transport with Piezoelectric Micro Diaphragm Pumps.

The automated transport of cells can enable far-reaching cell culture research. However, to date, such automated transport has been achieved with large pump systems that often come with long fluidic connections and a large power consumption. Improvement is possible with space- and energy-efficient piezoelectric micro diaphragm pumps, though a precondition for a successful use is to enable transport with little to no mechanical stress on the cell suspension. This study evaluates the impact of the microfluidic transport of cells with the piezoelectric micro diaphragm pump developed by our group. It includes the investigation of different actuation signals. Therewith, we aim to achieve optimal fluidic performance while maximizing the cell viability. The investigation of fluidic properties proves a similar performance with a hybrid actuation signal that is a rectangular waveform with sinusoidal flanks, compared to the fluidically optimal rectangular actuation. The comparison of the cell transport with three actuation signals, sinusoidal, rectangular, and hybrid actuation shows that the hybrid actuation causes less damage than the rectangular actuation. With a 5% reduction of the cell viability it causes similar strain to the transport with sinusoidal actuation. Piezoelectric micro diaphragm pumps with the fluidically efficient hybrid signal actuation are therefore an interesting option for integrable microfluidic workflows.

Smartphone-based colorimetric detection system for portable health tracking.

Colorimetric tests for at-home health monitoring became popular 50 years ago with the advent of the urinalysis test strips, due to their reduced costs, practicality, and ease of operation. However, developing digital systems that can interface these sensors in an efficient manner remains a challenge. Efforts have been put towards the development of portable optical readout systems, such as smartphones. However, their use in daily settings is still limited by their error-prone nature associated to optical noise from the ambient lighting, and their low sensitivity. Here, a smartphone application (Colourine) to readout colorimetric signals was developed on Android OS and tested on commercial urinalysis test strips for pH, proteins, and glucose detection. The novelty of this approach includes two features: a pre-calibration step where the user is asked to take a photo of the commercial reference chart, and a CIE-RGB-to-HSV color space transformation of the acquired data. These two elements allow the background noise given by environmental lighting to be minimized. The sensors were characterized in the ambient light range 100-400 lx, yielding a reliable output. Readouts were taken from urine strips in buffer solutions of pH (5.0-9.0 units), proteins (0-500 mg dL-1) and glucose (0-1000 mg dL-1), yielding a limit of detection (LOD) of 0.13 units (pH), 7.5 mg dL-1 (proteins) and 22 mg dL-1 (glucose), resulting in an average LOD decrease by about 2.8 fold compared to the visual method.

Automated, flow-based chemiluminescence microarray immunoassay for the rapid multiplex detection of IgG antibodies to SARS-CoV-2 in human serum and plasma (CoVRapid CL-MIA).

In the face of the COVID-19 pandemic, the need for rapid serological tests that allow multiplexing emerged, as antibody seropositivity can instruct about individual immunity after an infection with SARS-CoV-2 or after vaccination. As many commercial antibody tests are either time-consuming or tend to produce false negative or false positive results when only one antigen is considered, we developed an automated, flow-based chemiluminescence microarray immunoassay (CL-MIA) that allows for the detection of IgG antibodies to SARS-CoV-2 receptor-binding domain (RBD), spike protein (S1 fragment), and nucleocapsid protein (N) in human serum and plasma in less than 8 min. The CoVRapid CL-MIA was tested with a set of 65 SARS-CoV-2 serology positive or negative samples, resulting in 100% diagnostic specificity and 100% diagnostic sensitivity, thus even outcompeting commercial tests run on the same sample set. Additionally, the prospect of future quantitative assessments (i.e., quantifying the level of antibodies) was demonstrated. Due to the fully automated process, the test can easily be operated in hospitals, medical practices, or vaccination centers, offering a valuable tool for COVID-19 serosurveillance. Graphical abstract.

Precision 3D-Printed Cell Scaffolds Mimicking Native Tissue Composition and Mechanics.

Cellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell-ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell-ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two-photon stereolithography is adopted to print up to mm-sized high-precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein-based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two-pass printing or post-print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7-300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D-lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single-cell and tissue dynamics in response to defined mechanical and bio-molecular cues and is ultimately scalable to full organs.

A comparative study of photoplethysmogram and piezoelectric plethysmogram signals.

The Photoplethysmogram (PPG) signal is one of the most important vital signals in biomedical applications. The non-invasive property and the convenience in the acquisition of both PPG and Piezoelectric Plethysmogram (PZPG) signals are considered as powerful and accurate tools for biomedical diagnosing applications, such as oxygen saturation in blood, blood flow, and blood pressure measurements. In this paper, a number of features for PPG and PZPG signals (ex. first derivative, second derivative, area under the curve and the ratio of systolic area to the diastolic area) are acquired and compared. The results show that both systems are able to extract the pulse rate (PR) and pulse rate variability (PRV), accurately with an estimation error of less than 10%. The averaged standard deviation of the ratio of the systolic area to the diastolic area for the first derivative of PPG and PZPG signals was small with less than 0.49 and 0.69 for the PPG and PZPG, respectively. Statistical analysis techniques (such as cross-correlation, P-value test, and Bland Altman method) are performed to address the relation between the PPG and PZPG signals. All of these methods showed a strong relationship between the features of the two signals (i.e. PPG and PZPG). The correlation value is found to be 0.954 with a p-value of < 0.05. This opens possibilities for combining both the PPG and PZPG systems to extract more features that can be used in diagnosing cardiovascular diseases. Such a system can provide a possibility to reduce the number of devices connected to patients (especially in emergencies) by means of measuring simultaneously both signals (PZPG and PPG).

Monitoring tissue-level remodelling during inflammatory arthritis using a three-dimensional synovium-on-a-chip with non-invasive light scattering biosensing.

Rheumatoid arthritis is a chronic, systemic joint disease in which an autoimmune response translates into an inflammatory attack resulting in joint damage, disability and decreased quality of life. Despite recent introduction of therapeutic agents such as anti-TNFα, even the best current therapies fail to achieve disease remission in most arthritis patients. Therefore, research into the mechanisms governing the destructive inflammatory process in rheumatoid arthritis is of great importance and may reveal novel strategies for the therapeutic interventions. To gain deeper insight into its pathogensis, we have developed for the first time a three-dimensional synovium-on-a-chip system in order to monitor the onset and progression of inflammatory synovial tissue responses. In our study, patient-derived primary synovial organoids are cultivated on a single chip platform containing embedded organic-photodetector arrays for over a week in the absence and presence of tumor-necrosis-factor. Using a label-free and non-invasive optical light-scatter biosensing strategy inflammation-induced 3D tissue-level architectural changes were already detected after two days. We demonstrate that the integration of complex human synovial organ cultures in a lab-on-a-chip provides reproducible and reliable information on how systemic stress factors affect synovial tissue architectures.

Scleral Lens Sensor for Ocular Electrolyte Analysis.

The quantitative analysis of tear analytes in point-of-care settings can enable early diagnosis of ocular diseases. Here, a fluorescent scleral lens sensor is developed to quantitatively measure physiological levels of pH, Na+ , K+ , Ca2+ , Mg2+ , and Zn2+ ions. Benzenedicarboxylic acid, a pH probe, displays a sensitivity of 0.12 pH units within pH 7.0-8.0. Crown ether derivatives exhibit selectivity to Na+ and K+ ions within detection ranges of 0-100 and 0-50 mmol L-1 , and selectivities of 15.6 and 8.1 mmol L-1 , respectively. A 1,2 bis(o-aminophenoxy)ethane-N,N,-N',N'-tetraacetic-acid-based probe allows Ca2+ ion sensing with 0.02-0.05 mmol L-1 sensitivity within 0.50-1.25 mmol L-1 detection range. 5-Oxazolecarboxylic acid senses Mg2+ ions, exhibiting a sensitivity of 0.10-0.44 mmol L-1 within the range of 0.5-0.8 mmol L-1 . The N-(2-methoxyphenyl)iminodiacetate Zn2+ ion sensor has a sensitivity of 1 µmol L-1 within the range of 10-20 µmol L-1 . The fluorescent sensors are subsequently multiplexed in the concavities of an engraved scleral lens. A handheld ophthalmic readout device comprising light-emitting diodes (LEDs) and bandpass filters is fabricated to excite as well as read the scleral sensor. A smartphone camera application and an user interface are developed to deliver quantitative measurements with data deconvolution. The ophthalmic system enables the assessment of dry eye severity stages and the differentiation of its subtypes.

Label-Free High-Throughput Leukemia Detection by Holographic Microscopy.

Complete blood count and differentiation of leukocytes (DIFF) belong to the most frequently performed laboratory diagnostic tests. Here, a flow cytometry-based method for label-free DIFF of untouched leukocytes by digital holographic microscopy on the rich phase contrast of peripheral leukocyte images, using highly controlled 2D hydrodynamic focusing conditions is reported. Principal component analysis of morphological characteristics of the reconstructed images allows classification of nine leukocyte types, in addition to different types of leukemia and demonstrates disappearance of acute myeloid leukemia cells in remission. To exclude confounding effects, the classification strategy is tested by the analysis of 20 blinded clinical samples. Here, 70% of the specimens are correctly classified with further 20% classifications close to a correct diagnosis. Taken together, the findings indicate a broad clinical applicability of the cytometry method for automated and reagent-free diagnosis of hematological disorders.

Label-free, high-throughput detection of P. falciparum infection in sphered erythrocytes with digital holographic microscopy.

Effective malaria treatment requires rapid and accurate diagnosis of infecting species and actual parasitemia. Despite the recent success of rapid tests, the analysis of thick and thin blood smears remains the gold standard for routine malaria diagnosis in endemic areas. For non-endemic regions, sample preparation and analysis of blood smears are an issue due to low microscopy expertise and few cases of imported malaria. Automation of microscopy results could be beneficial to quickly confirm suspected infections in such conditions. Here, we present a label-free, high-throughput method for early malaria detection with the potential to reduce inter-observer variation by reducing sample preparation and analysis effort. We used differential digital holographic microscopy in combination with two-dimensional hydrodynamic focusing for the label-free detection of P. falciparum infection in sphered erythrocytes, with a parasitemia detection limit of 0.01%. Moreover, the achieved differentiation of P. falciparum ring-, trophozoite- and schizont life cycle stages in synchronized cultures demonstrates the potential for future discrimination of even malaria species.

Hybrid integration of scalable mechanical and magnetophoretic focusing for magnetic flow cytometry.

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.

One Binder to Bind Them All.

High quality binders, such as antibodies, are of critical importance for chemical sensing applications. With synthetic alternatives, such as molecularly imprinted polymers (MIPs), less sensor development time and higher stability of the binder can be achieved. In this feature paper, I will discuss the impact of synthetic binders from an industrial perspective and I will challenge the molecular imprinting community on the next step to leapfrog the current status quo of MIPs for (bio)sensing. Equally important, but often neglected as an effective chemical sensor, is a good match of transducer and MIP coating for a respective application. To demonstrate an application-driven development, a biosensing use case with surface-imprinted layers on piezoacoustic sensors is reported. Depending on the electrode pattern for the transducer, the strong mechanical coupling of the analyte with the MIP layer coated device allows the adoption of the sensitivity from cell mass to cell viability with complete reversibility.