Preparation Steps Research Articles

A plug-and-play, easy-to-manufacture fluidic accessory to significantly enhance the sensitivity of electrochemical immunoassays

Earlier access to patients’ biomarker status could transform disease management. However, gold-standard techniques such as enzyme-linked immunosorbent assays (ELISAs) are typically not deployed at the point-of-care due to their cumbersome instrumentation and complexity. Electrochemical immunosensors can be disruptive in this sector with their small size and lower cost but, without further modifications, the performance of these sensors in complex media (e.g., blood) has been limited. This paper presents a low-cost fluidic accessory fabricated using widely accessible materials and processes for boosting sensor sensitivity through confinement of the detection media next to the electrode surface. Liquid confinement first highlighted a spontaneous reaction between the pseudoreference electrode and ELISA detection substrate 3,3’,5,5’-tetramethylbenzidine (TMB) that decreases the amount of oxTMB available for detection. Different strategies are investigated to limit this and maximize reliability. Next, flow cell integration during the signal amplification step of sensor preparation was shown to substantially enhance the detection of cytokine interleukin-6 (IL-6) with the best sensitivity boost recorded for fresh human plasma (x7 increase compared to x5.8 in purified serum and x5.5 in PBS). The flow cell requires no specialized equipment and can be seamlessly integrated with commercial sensors, making an ideal companion for electrochemical signal enhancement.

Acoustofluidic separation of bacteria from platelets using tilted-angle standing surface acoustic wave

Separation of microscale objects, such as cells and bacteria, is an essential sample preparation step in various microfluidic applications in microbiology, chemistry, biology, and biomedicine. Microfluidic separation of bacteria from blood cells is highly in demand to prevent or investigate bacterial infectious diseases. Unlike red and white blood cells, platelets have a similar size distribution to that of bacteria, which makes bacteria separation from platelets technically challenging. Most previous techniques for microfluidic separation rely on size as a separation marker. These size-based separation methods are not applicable to separation of the same or similar-sized samples. Here, we propose an acoustofluidic method using tilted-angle standing surface acoustic wave (taSSAW) for label-free separation of the microscale objects of the same or similar size based on compressibility. From the experimental investigation with polymethylmethacrylate, polystyrene, and polycaprolactone microparticles of the same size, we found that less compressible objects migrated further when exposed to the taSSAW field formed within a microchannel. Based on the findings, we demonstrated that Escherichia coli (E. coli) can be separated from platelets because the taSSAW-induced acoustic radiation force acting on comparatively less compressible E. coli was greater than that applied to the platelets. Fluorescence microscopy, scanning electron microscope, hemocytometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and polymerase chain reaction were conducted to confirm the successful positive separation of bacteria from platelets with high purity of exceeding 96 % and recovery rate of exceeding 81 %.

Photogrammetry-Based Method for Measuring Volume Changes of Soil Specimens During Critical Phases of Triaxial Testing

Triaxial tests has been routinely used to measure the stress–strain relationship for geomaterials. During triaxial testing, many sources of errors that cannot be completely avoided but are often ignored or approximated using empirical equations. This paper presents a systematic investigation of soil volume change, volume strain nonuniformity, and cross-sectional calculation along the specimen during triaxial testing using a photogrammetry-based method. Consolidated drained triaxial tests were performed in which two parallel measurements were taken: (1) relative volume using the conventional triaxial testing and (2) absolute volume using the photogrammetry-based method. The difference in the observed volume and void ratio measurements between the two methods highlighted the importance of absolute soil volume over the relative volume method. The results revealed the effect of soil volume change in the interpretation of triaxial testing findings. Various preparation steps and procedures during triaxial testing have influenced the initial measured volume and thus caused deviation of the subsequent associated measurements. This method would present a quality control approach for different aspects of triaxial testing to improve the test simulations to accurately measure the soil behavior. The proposed method is important for more refined simulations and identification of setup-induced errors. Additional applications of the current research would allow correct determination of the stress path followed during triaxial testing, the critical-state soil mechanics parameters, and the stress–strain relationship, including deformation and strength parameters.

A Comprehensive Study on the Production and Characterization of Eco-Friendly Biodegradable Plastic Films from Dent Corn Starch

The increasing demand for eco-conscious and sustainable biomaterials has propelled the exploration of alternatives to petroleum-derived materials. This study delves into the utilization of dent corn (Zea mays L.) starch as a renewable resource for crafting biodegradable packaging solutions. The physicochemical properties of dent corn starch were meticulously assessed through a series of preparation steps, encompassing washing, cutting, grinding, drying, and pulverizing. Despite the inherent variability in dent corn sourced from local markets, the resulting starch exhibited commendable characteristics, including a pH of 7.02, moisture content of 6.50%, ash content of 0.20%, bulk density of 0.47 g/ml, gelatinization temperature of 61.2 ˚C, protein content of 2%, and a yield of 64%. Subsequently, employing the casting method, bioplastic films were synthesized using starch powder, water, and glycerol as a plasticizer. A comprehensive evaluation of the chemical, mechanical, solubility, and biodegradability properties of the films ensued. Remarkably, the films demonstrated notable values for tensile strength (2.58 MPa), elongation at break (24%), and film thickness (1.4 mm), alongside exhibiting low absorption rates in various media and remaining insoluble in solvents, even at elevated temperatures. Notably, the influence of glycerol content on the tensile strength and biodegradability of the films was elucidated through a least squares model, underscoring its pivotal role. These findings highlights the potential of dent corn starch as a viable alternative for the development of biodegradable packaging materials, despite the variability inherent in its sourcing from local markets. The study significantly contributes to ongoing endeavors aimed at fostering sustainability and reducing reliance on petroleum-derived materials, aligning with the escalating environmental consciousness.

Automation of 3D digital rolling circle amplification using a 3D-printed liquid handler

Automation of liquid handling is indispensable to improve throughput and reproducibility in biochemical assays. However, the incorporation of automated systems into laboratory workflows is often hindered by the high cost and complexity associated with building robotic liquid handlers. Here, we report a 3D-printed liquid handler based on a fluidic manifold, thereby obviating the need for complex robotic mechanisms. The fluidic manifold, termed a dispensing and aspirating (DA) device, comprises parallelized multi-pipette structures connected by distribution and aspiration channels, enabling the precise supply and removal of reagents, respectively. Leveraging the versatility of 3D printing, the DA device can be custom-designed and printed to fit specific applications. As a proof-of-principle, we engineered a 3D-printed liquid handler dedicated for 3D digital rolling circle amplification (4DRCA), an advanced biochemical assay involving multiple sample preparation steps such as antibody incubation, cell fixation, nucleic acid amplification, probe hybridization, and extensive washing. We demonstrate the efficacy of the 3D-printed liquid handler to automate the preparation of clinical samples for the simultaneous, in situ analysis of oncogenic protein and transcript markers in B-cell acute lymphoblastic leukemia cells using 4DRCA. This approach provides an effective and accessible solution for liquid handling automation, offering high throughput and reproducibility in biochemical assays.

An origami microfluidic paper device based on core-shell Cu@Cu2S@N-doped carbon hollow nanocubes

BackgroudThe global prevalence of diabetes mellitus, a serious chronic disease with fatal consequences for millions annually, is of utmost concern. The development of efficient and simple devices for monitoring glucose levels is of utmost significance in managing diabetes. The advancement of nanotechnology has resulted in the indispensable utilization of advanced nanomaterials in high-performance glucose sensors. Modulating the morphology and intricate composition of transition metals represents a viable approach to exploit their structure/function correlation, thereby achieving optimal electrocatalytic performance of the synthesized catalysts. ResultsHerein, a sensitive and rapid Cu-encapsulated Cu2S@nitrogen-doped carbon (Cu@Cu2S@N–C) hollow nanocubes-functionalized microfluidic paper-based analytical device (μ-PAD) was fabricated. Through a delicate sacrificial template/interface technique and thermal decomposition, inter-connected hollow networks were formed to boost the active sites, and the carbon shell was coated to protect Cu from being oxidation. For application, the constructed μ-PAD is used for glucose sensing utilizing an origami automated sample pretreatment system enabled by a simple application of strong alkaline solution on wax paper. Under optimal circumstances, the Cu@Cu2S@N–C electrochemical biosensor exhibits broad detection range of 2–7500 μM (R2 = 0.996) with low detection limit of 0.16 μM (S/N = 3) and high sensitivity of 1996 μA mM−1 cm−2. Additionally, the constructed μ-PAD also exhibited excellent selectivity, stability, and reproducibility. SignificanceBy rationally designing the double-shell hollow nanostructure and introducing Cu-encapsulated inner layer, the synthesized Cu@Cu2S@N–C hollow nanocubes show large specific surface area, short diffusion channels, and high stability. The proposed origami μ-PAD has been successfully applied to serum samples without any additional sample preparation steps for glucose determination, offering a new perspective for early nonenzymatic glucose diagnosis.

Effect of Cross-Sectional Designs of Different Ni-Ti Files on Shaping Ability After Repeated Usage in Simulated Root Canals.

Background and Objective: The study aimed was to compare the effect of cross-sectional designs of five rotary systems (One Curve, 2Shape, K3-i File, E3 Azure, Neolix), on the shaping ability of the files in simulated resin blocks under controlled conditions with five repeated use. The shaping ability of the files includes the following variables: (1) Longitudinal sectional area difference, (2) Canal wall surface area difference, (3) Canal volume difference, (4) SMI (structure material index) difference, (5) Cross-sectional area difference. Methods: Five rotary file systems: (1) One Curve, (2) 2Shape, (3) K3-i File, (4) E3 Azure, (5) Neolix, were tested in simulated J-shaped root canal resin blocks with a 45˚ angle of curvature. 10 files from each system, each one of the 10 files were used to prepare 5 resin blocks, named R1-R2-R3-R4-R5. All the used files from the 5 systems had tip size 25 diameter, and taper (6%), and length (25 mm). The first 4 rotary systems were used with a fixed speed (350 rpm), and fixed torque (3 Ncm), except for the fifth system which was used with a speed of (500 rpm), and torque (4 Ncm), Following the manufactural recommendation of each system. All the 250 resin blocks were prepared for Glide Path with manual reamers size 10, and 15, with a fixed working length 16 mm, then all the 250 samples were prepared with a Proglider rotary file from Dentsply (size 16 taper 3%), then finally were prepared with Edge Files (USA), with size 20 and taper 6%, reaching to the step of master preparation with the selected files of the 5 systems. Using a customized device for preparation to ensure fixed vertical force and to exclude any lateral force, preparation was done with EDTA solution as irrigation, with a fixed no. of 4 strokes for the first 5 systems and 9 strokes for the Neolix, the time was controlled by using a Metronome, each stroke duration was 6 seconds. Results: In the first, second, third, and fourth variables a statistically non-significant difference (P > 0.05) was found between One curve and 2Shape, a statistically significant difference (P < 0.05) was found between One Curve, K3-i File, E3 Azure, and Neolix. About the fifth variable which is the Cross-sectional area difference: the study compared the difference between the cross-section area of the canal at the point D8, comparing the cross-section area before and after preparation using ANOVA test and Post hoc test, a statistically significant difference (P < 0.05) was found between the Neolix and (2shape, K3 i-File, and E3 Azure). Conclusion: The cross-sectional design of the endodontic rotary instrument had an obvious effect on the shaping ability of the system. Keywords: Cross-sectional design, Shaping ability, Nickle Titanium rotary files (One Curve, 2Shape, K3-i File, E3 Azure, Neolix).

Surface and interface chemistry of bromine–methanol‐etched Cd0.9Zn0.1Te crystals

Chemical polishing or chemo‐mechanical polishing is crucial as a last step of surface preparation to remove the damaged layer and contaminants from the surface of CdZnTe crystals. The bromine–methanol solution is widely used for this purpose. However, bromine–methanol solution enriches the surface with Te and results in poor performance of CdZnTe crystals. In this study, the effect of the chemical polishing with 5% bromine–methanol solution on the surface and at the interface is investigated and it is demonstrated that etching duration strongly influences surface stoichiometry and interface contaminants. The evolution of the surface topography with etching and chemical changes are presented. It is shown that after 90 s etching/polishing, subsurface damage is removed and Te enrichment is minimum. Moreover, interface layer thickness is the smallest for 90 s etching duration. It is presented that further increase in the etching duration disturbs the surface stoichiometry and interface depth. It also calculated that 90 s of etching shows low interface barrier and symmetrical current–voltage curve.

Lipid loss and compositional change during preparation of liposomes by common biophysical methods.

Liposomes are widely used as model lipid membrane platforms in many fields, ranging from basic biophysical studies to drug delivery and biotechnology applications. Various methods exist to prepare liposomes, but common procedures include thin-film hydration followed by extrusion, freeze-thaw, and/or sonication. These procedures have the potential to produce liposomes at specific concentrations and membrane compositions, and researchers often assume that the concentration and composition of their liposomes are similar to, if not identical, to what would be expected if no lipid loss occurred during preparation. However, lipid loss and concomitant biasing of lipid composition can in principle occur at any preparation step due to nonideal mixing, lipid-surface interactions, etc. Here, we report a straightforward method using HPLC-ELSD to quantify the lipid concentration and membrane composition of liposomes, and apply that method to study the preparation of simple POPC/cholesterol liposomes. We examine many common steps in liposome formation, including vortexing during re-suspension, hydration of the lipid film, extrusion, freeze-thaw, sonication, and the percentage of cholesterol in the starting mixture. We found that the resuspension step can play an outsized role in determining the overall lipid loss (up to ~50% under seemingly rigorous procedures). The extrusion step yielded smaller lipid losses (~10-20%). Freeze-thaw and sonication could both be employed to improve lipid yields. Hydration times up to 60 minutes and increasing cholesterol concentrations up to 50 mole% had little influence on lipid recovery. Fortunately, even conditions with large lipid loss did not substantially influence the target membrane composition more than ~5% under the conditions we tested. From our results, we identify best practices for producing maximum levels of lipid recovery and minimal changes to lipid composition during liposome preparation protocols. We expect our results can be leveraged for improved preparation of model membranes by researchers in many fields.

Data-driven policy development of Municipalities Preparation steps for Integrating AI tools in the policymaking process

Data-driven policy development of Municipalities Preparation steps for Integrating AI tools in the policymaking process

Extracellular matrix of lung scaffolds submitted to different means of sterilization: a systematic review.

Chronic respiratory diseases often necessitate lung transplantation due to irreversible damage. Organ engineering offers hope through stem cell-based organ generation. However, the crucial sterilization step in scaffold preparation poses challenges. This study conducted a systematic review of studies that analysed the extracellular matrix (ECM) conditions of decellularised lungs subjected to different sterilisation processes. A search was performed for articles published in the PubMed, Web of Sciences, Scopus, and SciELO databases according to the PRISMA guidelines. Overall, five articles that presented positive results regarding the effectiveness of the sterilisation process were selected, some of which identified functional damage in the ECM. Was possible concluded that regardless of the type of agent used, physical or chemical, all of them demonstrated that sterilisation somehow harms the ECM. An ideal protocol has not been found to be fully effective in the sterilisation of pulmonary scaffolds for use in tissue and/or organ engineering.

Detecting Road Intersections from Crowdsourced Trajectory Data Based on Improved YOLOv5 Model

In recent years, the rapid development of autonomous driving and intelligent driver assistance has brought about urgent demands on high-precision road maps. However, traditional road map production methods mainly rely on professional survey technologies, such as remote sensing and mobile mapping, which suffer from high costs, object occlusions, and long updating cycles. In the era of ubiquitous mapping, crowdsourced trajectory data offer a new and low-cost data resource for the production and updating of high-precision road maps. Meanwhile, as key nodes in the transportation network, maintaining the currency and integrity of road intersection data is the primary task in enhancing map updates. In this paper, we propose a novel approach for detecting road intersections based on crowdsourced trajectory data by introducing an attention mechanism and modifying the loss function in the YOLOv5 model. The proposed method encompasses two key steps of training data preparation and improved YOLOv5s model construction. Multi-scale training processing is first adopted to prepare a rich and diverse sample dataset, including various kinds and different sizes of road intersections. Particularly to enhance the model’s detection performance, we inserted convolutional attention mechanism modules into the original YOLOv5 and integrated other alternative confidence loss functions and localization loss functions. The experimental results demonstrate that the improved YOLOv5 model achieves detection accuracy, precision, and recall rates as high as 97.46%, 99.57%, and 97.87%, respectively, outperforming other object detection models.

Does cervical root canal preparation influence the quality of endodontic treatment? A systematic review

Cervical preparation aims to remove dentin prominences located in the cervical region of the root canal. However, there is no consensus in the literature regarding the importance of this operative maneuver in endodontic treatment. The objective of this systematic review was to answer the question: Does cervical root canal preparation influence the quality of endodontic treatment? Material and methods: Electronic databases (Medline/PubMed, Lilacs, Scopus, Embase, Scientific Electronic Library Online – SciELO, and Central Register of Controlled Trials – Central) were searched until July 2023, without language or year restriction. Grey literature was also searched through Google scholar and OpenGrey repository. Only in vitro studies were included that evaluated the influence of cervical preparation on endodontic treatment. Relevant results were summarized and evaluated. The risk of bias was also assessed in the studies. Results: Initial screening of databases resulted in 235 studies, of which 80 were excluded for being duplicates. Of 155 eligible papers, twenty-nine studies met the inclusion criteria and were selected for full-text reading. Most of studies included in this review were qualified with low risk of bias. Five studies showed moderate risk. Conclusion: Based on the analyzed studies, it is possible to conclude that performing the cervical preparation facilitates the execution of the steps of odontometry and mechanical chemical preparation in clinical practice.

Esterification of Cyclic N6-Threonylcarbamoyladenosine During RNA Sample Preparation.

The continuous deciphering of crucial biological roles of RNA modifications and their involvement in various pathological conditions, together with their key roles in the use of RNA-based therapeutics, has reignited interest in studying the occurrence and identity of non-canonical ribonucleoside structures during the past years. Discovery and structural elucidation of new modified structures is usually achieved by combination of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) at the nucleoside level and stable isotope labeling experiments. This approach, however, has its pitfalls as demonstrated in the course of the present study: we structurally elucidated a new nucleoside structure that showed significant similarities to the family of (c)t6A modifications and was initially considered a genuine modification, but subsequently turned out to be an in vitro formed glycerol ester of t6A. This artifact is generated from ct6A during RNA hydrolysis upon addition of enzymes stored in glycerol containing buffers in a mildly alkaline milieu, and was moreover shown to undergo an intramolecular transesterification reaction. Our results demand for extra caution, not only in the discovery of new RNA modifications, but also with regard to the quantification of known modified structures, in particular chemically labile modifications, such as ct6A, that might suffer from exposure to putatively harmless reagents during the diverse steps of sample preparation.

Analytical Ultracentrifugation to Assess the Quality of LNP-mRNA Therapeutics.

The approval of safe and effective LNP-mRNA vaccines during the SARS-CoV-2 pandemic is catalyzing the development of the next generation of mRNA therapeutics. Proper characterization methods are crucial for assessing the quality and efficacy of these complex formulations. Here, we show that analytical ultracentrifugation (AUC) can measure, simultaneously and without any sample preparation step, the sedimentation coefficients of both the LNP-mRNA formulation and the mRNA molecules. This allows measuring several quality attributes, such as particle size distribution, encapsulation efficiency and density of the formulation. The technique can also be applied to study the stability of the formulation under stress conditions and different buffers.

Non-Enzymatic Glucose Sensors Composed of Polyaniline Nanofibers with High Electrochemical Performance.

The measurement of glucose concentration is a fundamental daily care for diabetes patients, and therefore, its detection with accuracy is of prime importance in the field of health care. In this study, the fabrication of an electrochemical sensor for glucose sensing was successfully designed. The electrode material was fabricated using polyaniline and systematically characterized using scanning electron microscopy, high-resolution transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and UV-visible spectroscopy. The polyaniline nanofiber-modified electrode showed excellent detection ability for glucose with a linear range of 10 μM to 1 mM and a detection limit of 10.6 μM. The stability of the same electrode was tested for 7 days. The electrode shows high sensitivity for glucose detection in the presence of interferences. The polyaniline-modified electrode does not affect the presence of interferences and has a low detection limit. It is also cost-effective and does not require complex sample preparation steps. This makes it a potential tool for glucose detection in pharmacy and medical diagnostics.

Enhanced selectivity for convenient extraction of acidic mycotoxins using a miniaturized centrifugal integrated cold-induced phase separation: Determination of fumonisins and ochratoxins in cereals as a proof-of-concept study

Starches-rich and protein-rich cereal samples commonly need tedious sample preparation steps before instrumental analysis. This study developed a miniaturized centrifugal integrated cold-induced phase separation (CIPS) method for convenient sample preparation. A small-sized centrifuge tube (2 mL) and a low-temperature centrifuge, both of which are easily accessible, make up the basic components of the system. Unlike conventional sample preparation methods that need a step-by-step extraction, enrichment, purification, and centrifugation, this centrifugal integrated CIPS method can be performed by a one-step combination protocol under a low-temperature centrifuge. As a proof-of-concept study, satisfactory recoveries and enrichment factors were demonstrated for the extraction of fumonisins and ochratoxins from cereals. A sensitive and selective quantification method was yielded by combining LC-HRMS using tSIM acquisition mode, with good linearity (R2 > 0.998), accuracy (82.9–106.5%), and precision (<13.4%). This strategy is convenient, low-cost, repeatable, and easy to semi-automate, further expanding the extraction potential for other acidic mycotoxins.

Recent advances in biosensors based on metal-oxide semiconductors system-integrated into bioelectronics

Metal-oxide semiconductors (MOSs) have emerged as pivotal components in technology related to biosensors and bioelectronics. Detecting biomarkers in sweat provides a glimpse into an individual's metabolism without the need for sample preparation or collection steps. The distinctive attributes of this biosensing technology position it as an appealing option for biomedical applications beyond the scope of diagnosis and healthcare monitoring. This review encapsulates ongoing developments of cutting-edge biosensors based on MOSs. Recent advances in MOS-based biosensors for human sweat analyses are reviewed. Also discussed is the progress in sweat-based biosensing technologies to detect and monitor diseases. Next, system integration of biosensors is demonstrated ultimately to ensure the accurate and reliable detection and analysis of target biomarkers beyond individual devices. Finally, the challenges and opportunities related to advanced biosensors and bioelectronics for biomedical applications are discussed.

Readsynth: short-read simulation for consideration of composition-biases in reduced metagenome sequencing approaches

BackgroundThe application of reduced metagenomic sequencing approaches holds promise as a middle ground between targeted amplicon sequencing and whole metagenome sequencing approaches but has not been widely adopted as a technique. A major barrier to adoption is the lack of read simulation software built to handle characteristic features of these novel approaches. Reduced metagenomic sequencing (RMS) produces unique patterns of fragmentation per genome that are sensitive to restriction enzyme choice, and the non-uniform size selection of these fragments may introduce novel challenges to taxonomic assignment as well as relative abundance estimates.ResultsThrough the development and application of simulation software, readsynth, we compare simulated metagenomic sequencing libraries with existing RMS data to assess the influence of multiple library preparation and sequencing steps on downstream analytical results. Based on read depth per position, readsynth achieved 0.79 Pearson’s correlation and 0.94 Spearman’s correlation to these benchmarks. Application of a novel estimation approach, fixed length taxonomic ratios, improved quantification accuracy of simulated human gut microbial communities when compared to estimates of mean or median coverage.ConclusionsWe investigate the possible strengths and weaknesses of applying the RMS technique to profiling microbial communities via simulations with readsynth. The choice of restriction enzymes and size selection steps in library prep are non-trivial decisions that bias downstream profiling and quantification. The simulations investigated in this study illustrate the possible limits of preparing metagenomic libraries with a reduced representation sequencing approach, but also allow for the development of strategies for producing and handling the sequence data produced by this promising application.

Injectable testosterone PLGA microsphere with different characteristics: effect of preparation method (paddle mixing versus microfluidic device)

The purpose of this study was to compare the characteristics of testosterone polylactic-co-glycolic (PLGA) microspheres prepared by a paddle mixer or microfluidics device. The comparison was conducted by not only in vitro evaluation but also in vivo evaluation which has not been reported up to date. We discovered that, among the steps in microsphere preparation, the solvent removal process strongly impacted drug content, particle size and surface morphology. Spectroscopic measurements suggested that molecular interactions and crystallinity of the drug incorporated in the microspheres differed. For the drug release profile, although both mixer- and microfluidics-prepared samples showed similar sustained release of the incorporated drug for approximately one month in vitro, they exhibited different plasma concentration profiles in vivo. Together, our findings show that the preparation process, especially the solvent removal process, may affect the physicochemical characteristics of testosterone PLGA microspheres, leading to different in vivo performance.