High Sample Throughput Research Articles

Artificial Intelligence Integration in Biochips: Enhancing Diagnostics and Precision Medicine

Abstract: The integration of biochips with AI opened up new possibilities and is expected to revolutionize smart healthcare tools within the next five years. The combination of miniaturized, multi- functional, rapid, high-throughput sample processing and sensing capabilities of biochips, with the computational data processing and predictive power of AI, allows medical professionals to collect and analyze vast amounts of data quickly and efficiently, leading to more accurate and timely diagnoses and prognostic evaluations. Biochips, as smart healthcare devices, offer continuous monitoring of patient symptoms. Integrated virtual assistants have the potential to send predictive feedback to users and healthcare practitioners, paving the way for personalized and predictive medicine. This review explores the current state-of-the-art biochip technologies including gene-chips, organ-on-a-chips, and neural implants, and the diagnostic and therapeutic utility of AI-assisted biochips in medical practices such as cancer, diabetes, infectious diseases and neurological disorders. Choosing the appropriate AI model for a specific biomedical application, and possible solutions to the current challenges are explored. Surveying advances in machine learning models for biochip functionality, this paper offers a review of biochips for the future of biomedicine, an essential guide for keeping up with trends in healthcare, while inspiring cross-disciplinary collaboration among biomedical engineering, medicine, and machine learning fields.

Assessment of greenness for solid phase microextraction techniques of sample preparation for flavors by AGREE Prep & SPMS tools

Assessing the greenness of sample preparation techniques is essential due to the use of various solvents, chemicals, reagents, sorbents, pH adjustments, and energetic inputs. Solid-Phase Microextraction (SPME) is preferred over traditional flavor extraction methods such as Simultaneous Distillation Extraction (SDE) and Solvent-Assisted Flavor Evaporation (SAFE) because of its sensitivity, efficiency, speed, versatility, and economy. SPME fibers coated with stationary phases can extract volatile and semi-volatile flavor compounds from food samples, which are then desorbed and analyzed by Gas Chromatography-Mass Spectrometry (GC–MS). This article provides an assessment of the greenness of 50 SPME techniques for flavor analysis using the AGREE Prep and Sample Preparation Metric of Sustainability (SPMS) tools. This evaluation offers valuable insights into the environmental impact and sustainability of SPME as sample preparation techniques for flavour analysis. As per AGREE Prep, method 34 was found to be environmental friendly with score of 0.66, attributed to safe solvents, minimized waste, high sample throughput, and low energy consumption where as method 7 with SPMS scores of 7.05 is more sustainable due to miniaturization, fewer steps, and low energy use.

Mapping soil VOCs using three green sample extraction techniques and GC–MS

Sample preparation is a very crucial step prior to sample analysis. Three green adsorptive sample preparation micro-extraction techniques were investigated (smart solid-phase microextraction (SPME), high-capacity sorptive extraction (HiSorb), and stir bar sorptive extraction (SBSE)) for the determination of volatile organic compounds (VOCs) emitted from vineyard soil samples after method optimization. More specifically, the 95 μm Carbon Wide Range/Polydimethylsiloxane (CWR/PDMS) smart SPME fiber, CWR/PDMS HiSorb, and PDMS Twister micro-extraction tools were compared for their extraction capabilities. The headspace (HS)-SPME-gas chromatography-mass spectrometry (GC–MS) method was proved better for the extraction of VOCs from the vineyard soils due to shorter exposure time (45 min), its automation (use of a fully automated system compared to the other two), and the high sample throughput analysis. The validation of the HS-SPME-GC–MS method showed good linearity (R2 ≥ 0.9951) and limit of detection (LOD), whereas the limit of quantitation (LOQ) ranged from 0.04-3.65 μg/kg. Also, the repeatability and reproducibility of the results showed a relative standard deviation (RSD) lower than 4.73 %. A total number of 47 soil-derived VOCs were identified through the validated HS-SPME-GC–MS method, and 9 targeted VOCs were quantified with total average concentrations ranging from 0.14-28.07 µg/kg. Smart SPME fiber proved beneficial over the two other tools, highlighting the aspects of green chemistry.

Humic acid-coated paper: An affordable mixed-mode weak-cation exchanger for isolating cocaine, codeine, and methadone from saliva samples for their direct infusion mass spectrometric analysis.

The use of natural materials as precursors for synthesizing sorptive phases can reduce the environmental impact of the sample preparation step. In this article, two natural materials, i.e., cellulose and humic acids, are combined, resulting in a porous and flat sorptive phase that combines hydrophobic and cation exchange interactions. The simple preparation, with low reagent consumption, and the high production capacity make this process affordable. The performance of the humic acid-coated paper has been studied towards three basic drugs (cocaine, codeine, and methadone), and it has allowed the determination of the targets in saliva by direct infusion tandem mass spectrometry. The simultaneous extraction of many samples and the rapid analysis (2 min) provide a high sample throughput. The resulting method has been validated following the International Conference on Harmonization (ICH) M10 validation guide. Working at the optimum conditions, the linear range was established from 5 to 500 μg·L-1 and the detection limits were established at 1.5 μg·L-1 for all the analytes. At the LOQ, the inter-day precision and intra-day precision, expressed as relative standard deviation, were better than 13.6% and 17.9%, respectively. For low/mid/high quality controls, the inter-day precision and intra-day precision were better than 11.8% and 9.1%, respectively. The accuracy, calculated as relative recovery, was in the range of 83.8-106.1%. Several samples from patients under treatment with codeine-containing prescriptions were finally analyzed.

B-154 Development and Evaluation of an Automated High-Throughput Magnetic Bead-Based pre-treatment Platform for Lipid-Soluble Vitamins in Clinical Mass Spectrometry

Abstract Background Mass spectrometry has become a critical tool in clinical diagnostics due to its unparalleled sensitivity, specificity, and ability for multiplex analysis, redefining standards in small molecule detection. However, its efficacy is limited by complex sample preparation requirements, such as purification and enrichment. Traditional pre-treatment methods often involve labor-intensive, error-prone procedures that can dilute samples, thereby necessitating enhanced sensitivity in mass spectrometry. Addressing these challenges, we developed an automated, high-throughput pre-treatment platform using magnetic bead technology to streamline the preparation of small molecules for analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study assesses the platform's effectiveness and reliability using lipid-soluble vitamins as a test case. Methods We developed a magnetic bead-based extraction process for lipid-soluble vitamins (Vitamins A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, E, and K1) from clinical serum samples, utilizing Zybio's EXM3000 for automation. Protein-bound vitamins were dissociated using methanol and isopropanol, followed by magnetic bead extraction. The extracted vitamins underwent LC-MS/MS analysis. Comparative analysis with traditional liquid-liquid extraction methods, using 20 clinical samples and commercially available kits, evaluated this novel approach's analytical efficacy. Results The method showed excellent linearity (r > 0.99) for the target compounds. The imprecision ranged from 1.5% to 9.3%, and the accuracy (recovery rate) was between 91.5% and 113.4%. The limit of quantification, repeated six times, had a deviation of -8.5% to 13% and a CV of 1.5%-6.4%, meeting industry standards and EP document requirements. Comparisons with commercial kits in 20 clinical samples showed deviations within ±15%, and the 95% confidence interval for expected bias met the testing requirements. The method also demonstrated high correlation with the commercial kits, as evidenced by regression equations for Vitamin A: y = 0.9732x - 1.7985, 25-hydroxyvitamin D2: y = 1.0163x - 0.0055, 25-hydroxyvitamin D3: y = 1.0597x - 0.7968, Vitamin E: y = 1.0569x - 0.2917, and Vitamin K1: y = 1.0055x - 0.0039. Conclusions This study showed excellent performance of a novel automated high-throughput magnetic bead sample preparation platform for small molecules in clinical mass spectrometry. This technology overcomes the complexities associated with sample pre-treatment in clinical mass spectrometry and holds broad clinical application prospects in mass spectrometric analysis.

One-step TaqMan® RT-qPCR detection of sugar beet-infecting poleroviruses in Myzus persicae from yellow water pan traps opens up new possibilities for early risk assessment of virus yellows disease

Virus yellows disease (VY) is a major threat to sugar beet production in Europe. Beet chlorosis virus (BChV) and beet mild yellowing virus (BMYV) are of particular economic importance and are both persistently transmitted by the aphid vector Myzus persicae. As part of integrated pest management strategies, M. persicae influx into sugar beet fields is recorded weekly using yellow water pan traps. To date, only ELISA and RT-PCR assays have been described for BChV and BMYV detection in individual aphids.In this study, we describe for the first time two one-step TaqMan® RT-qPCR assays designed for the specific detection of BChV and BMYV in M. persicae after 7d incubation in water pan trap medium. Both viruses were reproducibly detected in individual aphids. After 7d incubation in trap medium, both viruses were reproducibly detected in individual aphids, as well as in one viruliferous aphid in a pool of 99 non-viruliferous aphids. Significant correlations can be shown between different mixing ratios of viruliferous to non-viruliferous aphids and Ct values of total RNA templates, allowing the percentage of viruliferous aphids in yellow water pan traps to be estimated using a standard curve.The described methodology provides a high sensitivity combined with a high sample throughput and can be used, after evaluation in the field, for practical monitoring, risk modelling and development of decision support systems for VY.

Essential Fluidics for a Flow Cytometer.

Flow cytometry is an inherently fluidic process that flows particles on a one-by-one basis through a sensing region to discretely measure their optical and physical properties. It can be used to analyze particles ranging in size from nanoparticles to whole organisms (e.g., zebrafish). It has particular value for blood analysis, and thus most instruments are fluidically optimized for particles that are comparable in size to a typical blood cell. The principles of fluid dynamics allow for particles of such size to be precisely positioned in flow as they pass through sensing regions that are tens of microns in length at linear velocities of meters per second. Such fluidic systems enable discrete analysis of cell-sized particles at rates approaching 100 kHz. For larger particles, the principles of fluidics greatly reduce the achievable rates, but such high rates of data acquisition for cell-sized particles allow rapid collection of information on many thousands to millions of cells and provides for research and clinical measurements of both rare and common cell populations with a high degree of statistical confidence. Additionally, flow cytometers can accurately count particles via the use of volumetric sample delivery and can be coupled with high-throughput sampling technologies to greatly increase the rate at which independent samples can be delivered to the system. Due to the combination of high analysis rates, sensitive multiparameter measurements, high-throughput sampling, and accurate counting, flow cytometry analysis is the gold standard for many critical applications in clinical, research, pharmaceutical, and environmental areas. Beyond the power of flow cytometry as an analytical technique, the fluidic pathway can be coupled with a sorting mechanism to collect particles based on desired properties. We present an overview of fluidic systems that enable flow cytometry-based analysis and sorting. We introduce historical approaches, explanations of commonly implemented fluidics, and brief discussions of potential future fluidics where appropriate. © 2024 Wiley Periodicals LLC.

Workflow improvement and financial gain after integration of high-throughput sample processing system with flow cytometer in a high-volume pathology laboratory: Results from a prospective comparative study using Lean principles

BackgroundHighly efficient clinical laboratories are essential for monitoring many human illnesses. Ampath Laboratory Services, the largest pathology lab in South Africa, analyzes large numbers of peripheral blood samples for CD4 levels yearly. ObjectiveTo assess productivity and quality of a newer integrated automated solution, the BD FACSDuet™ Sample Preparation System/BD FACSLyric™ Flow Cytometer using conventional assessment methods and Lean concepts. Materials and methodsThis prospective study compared the performance of the BD FACS™ Sample Preparation Assistant [SPA] III and BD FACSCanto™ II Flow Cytometer with the newly introduced integrated system (BD FACSDuet™/BD FACSLyric™). They were validated for accuracy, precision, and external quality assessment. Process mapping and Lean assessment helped identify steps leading to waste. An economic model was developed to characterize workflow and economic impact associated with total daily hands-on time, processing time, and reworks. ResultsStrong linear correlation was present between both systems. Precision and accuracy studies revealed that all coefficient of variation (CV)% values were below 20 % of allowable limits. External proficiency assessments were within limits. The fully automated workflow of BD FACSDuet™/BD FACSLyric™ permitted better consistency with significantly shorter processing time and batch processing and reduced operator interventions. Lean assessment identified defects with motion, over-processing, waiting, and non-utilized talent. Significant reductions in hands-on and total daily processing time that could increase daily specimen testing efficiency and fewer reworks were associated with the BD FACSDuet™/BD FACSLyric™. Lean improvements translated to significant economic savings associated with operator costs and unnecessary reagent consumption. ConclusionBD FACSDuet™/BD FACSLyric™ is an accurate, reliable, and cost-effective fully automated system for high-volume flow cytometry labs that perform T-cell enumeration using a single-platform and single-tube approach.

A Simple Eco-Friendly HPLC-PDA Method for the Simultaneous Determination of Paclitaxel and Seliciclib in Plasma Samples for Assessing Their Pharmacodynamics and Pharmacokinetics in Combination Therapy for Uterine Sarcoma.

Background/Objectives: Uterine sarcoma, a rare cancer originating in the smooth muscle of the uterus, exhibits high rates of recurrence and metastasis. It represents one of the most challenging types of cancer due to its chemorefractory nature, showing little response to conventional chemotherapy methods and displaying a relative survival rate of 30-40%. A potentially promising approach for treating uterine sarcoma involves combination therapy with paclitaxel (PAC), a microtubule-targeting agent, and seliciclib (SEL), a cyclin-dependent kinase inhibitor. SEL has been identified as a drug that can enhance the effectiveness of PAC through synergistic effects. To further refine this treatment strategy, an efficient analytical tool capable of simultaneously measuring the concentrations of PAC and SEL in blood plasma is needed. This tool would make it easier to study the pharmacokinetic interactions of potential drugs and assist in monitoring therapy when administering this combination treatment. Regrettably, a method meeting these specific requirements has not been documented in the existing literature. Methods: This article introduces the first HPLC technique employing a PDA detector to concurrently measure PAC and SEL levels in plasma. The methodology underwent validation in accordance with the ICH standards for validating bioanalytical methods. Results: The method exhibited linearity in the concentrations ranging from 0.8 to 100 µg mL-1 for both PAC and SEL. The limits of quantification were determined and found to be 1.34 and 1.25 µg mL-1 for PAC and SEL, respectively. All the other validation criteria conformed to the ICH validation standards. The HPLC-PDA method was successfully employed to quantify both PAC and SEL in plasma samples with a high level of reliability (in terms of accuracy and precision). The eco-friendliness of the approach was verified using three thorough assessments. This technique serves as a valuable asset in establishing the correct dosage and administration schedule for the combined treatment involving PAC and SEL, ensuring the desired therapeutic effects and safety in managing uterine sarcoma. Conclusions: The proposed HPLC-PDA method is the first reliable and eco-friendly method developed to simultaneously determine PAC and SEL in high-throughput plasma samples in clinical laboratories.

Thiol-functionalized MOF modified 3D printed monolithic microextraction array for analysis of trace Cd and Pb in human urine

Controlling the position, size, and shape of pores is a limitation of traditional monolithic preparation methods. The application of 3D printing technology offers high customizability, allowing the precise printing of pore positions, sizes, and shapes according to the designer's 3D model. Herein, by using Projection Microstereolithography (PμSL), we prepared a 3D-printed monolithic array with post-modification of thiol-functionalized metal-organic framework (MOF), and combined it with inductively coupled plasma mass spectrometry (ICP-MS) for the online analysis of trace Cd and Pb in human urine. To achieve array monolithic microextraction, six 3D-printed monolithic columns were modified with thiol-functionalized MOF-808 (MOF-808-SH), and were then assembled in the 3D printed extraction device incorporating gas valve and scaffold. The MOF-808-SH modified 3D-printed monolithic column exhibits excellent extraction performance to Cd2+ and Pb2+ due to rich active adsorption sites and hierarchical porous structure, and has long life span (>100 reused times). Under the optimized conditions, the limits of detection (LODs) are 3.5 and 17.6 ng L−1 for Cd2+ and Pb2+, respectively, with the relative standard deviations of 4.9 % and 8.2 % (0.1 μg L−1, n = 7), and the sample throughput is 11 h−1. To validate the accuracy of the method, the method was used to determine Cd and Pb in Certified Reference Materials of freeze-dried human urine, the determined results agree well with the certified values. This method was also successfully applied to the determination of trace Cd and Pb in real human urine samples. The developed method offers low LODs, robust anti-interference capability, high sample throughput, long reuse cycles, and automation analysis, showing great potential for the analysis of trace heavy metals in biological samples.

Intrinsic interlayer shear strength of graphite

Graphite holds significant values in the energy and electronics industries due to its unique properties. As a quintessential example of highly anisotropic materials, the shear strength measures one of its most fundamental mechanical properties. However, the lack of ideal materials and testing methods has led to a wide dispersion in the reported values. To address this issue, we utilized epitaxially grown single-crystal graphite and developed a high-throughput sample preparation method, along with a novel loading technique in this work. The intrinsic shear strength of AB-stacked graphite was determined to be τs = 62 MPa, by excluding the size effect in measurements. The results are further compared to highly oriented pyrolytic graphite specimens processed down to nanoscale thickness, highlighting the adverse impact of twisted single-crystalline interfaces between the graphitic layers. Additionally, we observed a distinctive failure mechanism with continuous and uniform cascade plastic slips across the thickness of graphite samples, which corresponds to an interlayer shear strength approaching τs. The intrinsic shear strength characterized in our work sets an upper limit for the interlayer shear resistance of graphite. The experimental procedure for measuring shear strength can be applied to other van der Waals materials.

Sample-Specific MS/MS Methods in High-Throughput Mass Spectrometry.

The drug discovery process increasingly relies on high-throughput sample analysis to accelerate the identification of viable drug candidates. Recently, chromatographic-free high-throughput mass spectrometry (HT-MS) technologies have emerged, significantly increasing sample readout speed and enabling the analysis of large sample sets. These HT-MS platforms continuously acquire data from various samples into a single data file, presenting challenges in applying distinctive data acquisition methods to specific samples. This study introduces a novel approach that integrates real-time sample loading status to activate sample-specific MS/MS data acquisition methods on the high-throughput acoustic ejection mass spectrometry platform. Effective method switching and high signal reproducibility were demonstrated across different data acquisition window durations in multiple reaction monitoring (MRM), high-resolution MRM (MRMHR), and information-dependent acquisition modes. This advancement provides a user-friendly and robust solution to the method-setting challenges of HT-MS, expanding the implementation of HT-MS platforms in drug discovery and other high-throughput analytical applications.

Mid‐Infrared Single‐Photon Compressive Spectroscopy

AbstractSensitive mid‐infrared (MIR) spectroscopy plays an indispensable role in various photon‐starved conditions. However, the detection sensitivity of conventional MIR spectrometers is severely limited by excessive noises of the involved infrared sensors, especially for multi‐pixel arrays in parallel spectral acquisition. Here, an ultra‐sensitive MIR single‐pixel spectrometer is devised and implemented, which relies on high‐fidelity spectral upconversion and wavelength‐encoding compressive measurement. Specifically, a MIR nanophotonic supercontinuum from 3.1 to 3.9 µm is nonlinearly converted to the NIR band via synchronous chirped‐pulse pumping, which facilitates both the precise spectral mapping and sensitive upconversion detection. The upconverted signal is then spatially dispersed onto a programmable digital micromirror device, before being registered by a single‐element silicon detector. Consequently, the spectral information can be deciphered from the correlation between encoded patterns and recorded measurements, which results in a spectral resolution of 0.5 under an illumination flux down to 0.01 photons nm–1 pulse–1. Moreover, faithful reconstructions at sub‐Nyquist sampling rates are demonstrated using the compressive sensing algorithm, which leads to a 95% reduction in data acquisition time. The presented single‐pixel computational spectrometer features wavelength multiplexing, high throughput, and efficient sampling, which thus paves a new way for sensitive and fast spectroscopic analysis at the single‐photon level.

A high throughput facility for the RF characterisation Of planar superconducting thin films

Abstract Accelerator laboratories worldwide are researching copper radio frequency (RF) cavities coated with superconducting thin films to exceed the limits of bulk niobium. The development and RF testing of thin films on small planar samples is vital before cavity depositions. A team at Daresbury Laboratory have developed a cost-effective facility using a novel 7.8 GHz Choke Cavity for the RF characterisation of planar samples. RF chokes ensure that no electrical contact is required between the sample and the cavity. The main advantages are: a simple sample design (90 − 130 mm diameter disk with no sample-cavity welding) and easy operation using a LHe-free cryostat. This enables high sample throughput, with up to 3 sample tests per week, making the facility suitable for quick, systematic scanning of deposition parameters. With the sample thermally and physically isolated from the test cavity, it is possible to measure the average surface resistance, R s, directly using an RF-DC compensation method. Facility commissioning has been performed with bulk and thin film niobium samples. These tests have demonstrated the ability to measure R s at temperatures in the range 4 − 20 K and sample peak magnetic fields up to 3 mT. The minimum resolvable R s is 0.5 μΩ with typical uncertainties of 9 − 15%. The design, operation and commissioning of this facility is reported in this paper.

Machine-Learning-Accelerated DFT Conformal Sampling of Catalytic Processes.

Computational modeling of catalytic processes at gas/solid interfaces plays an increasingly important role in chemistry, enabling accelerated materials and process optimization and rational design. However, efficiency, accuracy, thoroughness, and throughput must be enhanced to maximize its practical impact. By combining interpolation of DFT energetics via highly accurate Machine-Learning Potentials with conformal techniques for building the training database, we present here an original approach (that we name Conformal Sampling of Catalytic Processes, CSCP), to accelerate and achieve an accurate and thorough sampling of novel systems by exporting existing information on a worked-out case. We use methanol decomposition (of interest in the field of hydrogen production and storage) as a test catalytic reaction. Starting from worked-out Pt-based systems, we show that after only two iterations of active-learning CSCP is able to provide reaction energy diagrams for a set of 7 diverse systems (Pd, Ni, Au, Ag, Cu, Co, Fe) leading to DFT-accuracy-level predictions. Cases exhibiting a change in adsorption sites and mechanisms are also successfully reproduced as tests of catalytic path modification. The CSCP approach thus offers itself as an operative tool to fully take advantage of accumulated information to achieve high-throughput sampling of catalytic processes.

A high-throughput sample preparation approach for the determination of nonsteroidal anti-inflammatory drugs in urine samples through membrane-based microextraction employing a green supramolecular solvent

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of drugs with antipyretic, analgesic, and anti-inflammatory effects widely used worldwide. This study aimed at developing an environmentally friendly and high-throughput sample preparation method for the determination of six NSAIDs in urine samples followed by high-performance liquid chromatography coupled to diode-array detection (HPLC-DAD). The experimental workflow consisted of hollow-fiber microporous membrane liquid-liquid extraction (HF-MMLLE) using a supramolecular solvent immobilized into the porous of polypropylene hollow membranes (1 cm length). These membranes were attached to a 96-well plate system to permit multiple extractions simultaneously. Under the optimized conditions, limits of detection (LODs) and limits of quantification (LOQs) ranged from 6.06 to 30.3 µg/L and from 20 to 100 µg/L, respectively. The linear ranges varied from 20 to 1000 µg/L for indomethacin and diclofenac, from 50 to 1000 µg/L for ketoprofen and ibuprofen, and from 100 to 1000 µg/L for naproxen and fenoprofen. The coefficients of determination (R²) ranged from 0.9735 to 0.9997. Intraday precision ranged from 6.13 to 20.11 %, interday precision varied from 2.87 to 20.72 %, and accuracy ranged from 92.49 to 126.89 %. Moreover, the recently proposed Sample Preparation Metric of Sustainability (SPMS) was employed to evaluate the greenness of the sample preparation technique resulting in a score of 8.0, which is highly satisfactory according to the concepts of green analytical chemistry.

Machine Learning for High-Throughput Configuration Sampling of Li−La−Ti−O Disordered Solid-State Electrolyte

Machine Learning for High-Throughput Configuration Sampling of Li−La−Ti−O Disordered Solid-State Electrolyte

Biolayer interferometry for adeno-associated virus capsid titer measurement and applications to upstream and downstream process development

Faster and more accurate analytical methods are needed to support the advancement of recombinant adeno-associated virus (rAAV) production systems. Recently, bio-layer interferometry (BLI) has been developed for high-throughput AAV capsid titer measurement by functionalization of the AAVX ligand onto biosensor probes (AAVX-BLI). In this work, an AAVX-BLI method was evaluated using Octet® AAVX biosensors across four rAAV serotypes (rAAV2, 5, 8, and 9) and applied in an upstream and downstream processing context. AAVX-BLI measured capsid titer across a wide concentration range (1×1010 – 1×1012 capsids/mL) for different rAAV serotypes and sample backgrounds with reduced measurement variance and error compared to an enzyme-linked immunosorbent assay (ELISA) method. Biosensors were regenerated for repeated use, with lysate samples showing reduced regeneration capacity compared to purified and supernatant samples. The AAVX-BLI method was applied in a transfection optimization study where direct capsid titer measurement of culture supernatants generated a representative response surface for total vector genome (VG) titer. For rAAV purification, AAVX-BLI was used to measure dynamic binding capacity with POROS™ CaptureSelect™ AAVX affinity chromatography, showing resin breakthrough dependence on operating flow rate. Measurement accuracy, serotype and sample background flexibility, and high sample throughput make AAVX-BLI an attractive alternative to other capsid titer measurement techniques.

A Zwitterionic Detergent and Catalyst-Based Single-Cell Proteomics Using a Loss-Free Microhole-Collection Disc.

Recent advances in single-cell proteomics have solved many bottlenecks, such as throughput, sample recovery, and scalability via nanoscale sample handling. In this study, we aimed for a sensitive mass spectrometry (MS) analysis capable of handling single cells with a conventional mass spectrometry workflow without additional equipment. We achieved seamless cell lysis and TMT labeling in a micro-HOLe Disc (microHOLD) by developing a mass-compatible single solution based on a zwitterionic detergent and a catalyst for single-cell lysis and tandem mass tag labeling without a heat incubation step. This method was developed to avoid peptide loss by surface adsorption and buffer or tube changes by collecting tandem mass tag-labeled peptide through microholes placed in the liquid chromatography injection vials in a single solution. We successfully applied the microHOLD single-cell proteomics method for the analysis of proteome reprogramming in hormone-sensitive prostate cells to develop castration-resistant prostate cancer cells. This novel single-cell proteomics method is not limited by cutting-edge nanovolume handling equipment and achieves high throughput and ultrasensitive proteomics analysis of limited samples, such as single cells.

Quantitative assessment of reef foraminifera community from metabarcoding data.

Describing living community compositions is essential to monitor ecosystems in a rapidly changing world, but it is challenging to produce fast and accurate depiction of ecosystems due to methodological limitations. Morphological methods provide absolute abundances with limited throughput, whereas metabarcoding provides relative abundances of genes that may not correctly represent living communities from environmental DNA assessed with morphological methods. However, it has the potential to deliver fast descriptions of living communities provided that it is interpreted with validated species-specific calibrations and reference databases. Here, we developed a quantitative approach to retrieve from metabarcoding data the assemblages of living large benthic foraminifera (LBF), photosymbiotic calcifying protists, from Indonesian coral reefs that are under increasing anthropogenic pressure. To depict the diversity, we calculated taxon-specific correction factors to reduce biological biases by comparing surface area, biovolume and calcite volume, and the number of mitochondrial gene copies in seven common LBF species. To validate the approach, we compared calibrated datasets of morphological communities from mock samples with bulk reef sediment; both sample types were metabarcoded. The calibration of the data significantly improved the estimations of genus relative abundance, with a difference of ±5% on average, allowing for comparison of past morphological datasets with future molecular ones. Our results also highlight the application of our quantitative approach to support reef monitoring operations by capturing fine-scale processes, such as seasonal and pollution-driven dynamics, that require high-throughput sampling treatment.