Low Sample Volume Research Articles

Abstract P20: Unveiling the Influence of Tumor Microenvironment and Spatial Heterogeneity on Temozolomide Resistance in Glioblastoma using an Advanced Human in vitro Model of the Blood-brain Barrier and Glioblastoma

Abstract Glioblastoma (GBM) is the most common primary malignant brain cancer in adults, with a dismal prognosis of 15 months. This is despite decades of research and improvements in surgery and radiotherapy. Temozolomide (TMZ) is the only first-in-line chemotherapeutic; however, resistance is frequent and multifactorial. While many molecular and genetic factors have been linked to TMZ resistance, the role of the solid tumor morphology and the tumor microenvironment (TME), particularly the blood-brain barrier (BBB), is unknown. Complex 3D in vitro models are able to recapitulate aspects of clinical disease such as the tumor microenvironment, such that research into the role of the TME and how it affects tumor biology and therapy resistance can be studied more easily and accurately. We have developed a complex 3D in vitro model for GBM and its surrounding BBB, that recapitulates important clinical features such as a dense tumor core with tumor cells that invade along the perivascular space; and a perfusable BBB with a physiological permeability with morphology that is altered in the presence of a tumor spheroid. We demonstrate that TMZ sensitivity decreases with increasing cancer cell spatial organization, and that the BBB can contribute to TMZ resistance. Proteomic analysis of cultured microtissues using low volume sample workflows revealed proteins linked to aggressive tumors and TMZ resistance. Notably, proteomic analysis revealed a potential role for cell cycle proteins in TMZ resistance. Preliminary data with tumor spheroids formed by patient derived GBM progenitor cells revealed a similar trend of an increase in proteins associated with tumor aggressiveness upon co-culture with a BBB. The data demonstrate the utility of combining complex physiological in vitro models and next generation mass spectrometry for interrogating the tumour microenvironment, tumor biology and accurate in vitro therapy validation. Citation Format: Maxine SY Lam, Joey JY Aw, Damien Tan, Ragavi Vijayakumar, Hui Yi Grace Lim, Swathi Yada, Qing You Pang, Nick Barker, Carol Tang, Beng Ti Ang, Radoslaw M. Sobota, Andrea Pavesi. Unveiling the Influence of Tumor Microenvironment and Spatial Heterogeneity on Temozolomide Resistance in Glioblastoma using an Advanced Human in vitro Model of the Blood-brain Barrier and Glioblastoma [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P20.

Comparative analysis of human tear fluid and aqueous humor proteomes

PurposeTechnological advancements allowing for the analysis of low-volume samples have led to the investigation of human tear fluid and aqueous humor (AH) as potential biomarker sources. However, acquiring AH samples poses significant challenges, making human tear fluid a more accessible alternative. This study aims to compare the protein compositions of these two biofluids to evaluate their suitability for biomarker discovery. MethodsPaired tear and AH samples were collected from 20 patients undergoing cataract surgery. Tear samples were collected using Schirmer strips prior to surgery, and AH samples were collected from the anterior chamber immediately after corneal incision. Proteins were extracted and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). ResultsA total of 481 proteins were identified in greater than 50% of the tear samples, and 191 proteins were detected in greater than 50% of the AH samples. Of these proteins, 82 were found to be common between the two biofluids, with ALB, LTF, TF, LCN1, and IGKC being the most abundant. ConclusionAlthough tear fluid and the AH are functionally independent and physically separated, many of the proteins detected in AH were also detected in tears. This direct comparison of the proteomic content of tear fluid and AH may aid in further investigation of tear fluid as a source of readily accessible biomarkers for various human diseases.

Neonatal Blood Transfusion in Africa; Towards Optimizing Scarce Resource

Neonatal transfusion is a common practice in Africa, where many infants are born with low birth weight and require blood transfusions. The use of paediatric packs for neonatal blood transfusions has been recommended. However, the optimal use of these packs in Africa is not clearly understood by some healthcare practitioners. This review aims to provide insights into the optimal use of paediatric packs in neonatal transfusion in Africa.The World Health Organization (WHO) recommends restrictive transfusion practices for paediatric patients, including neonates, based on expert opinion. Transfusion triggers in neonates are controversial and mainly based on expert clinical opinion, although recent randomized controlled trials of ‘liberal’ versus ‘restrictive’ red cell transfusion policies in very low birth weight preterm babies are starting to influence clinical guidelines. Many neonatal red cell transfusions are given to replace losses from frequent blood sampling. This can be reduced by avoiding non-essential tests, using low-volume sample tubes, validated near patient testing, micro-techniques in the laboratory, and non-invasive monitoring where possible.The use of CMV-seronegative blood products for transfusion in low birth weight neonates is not deemed necessary. The primary rationale is that the risk of transfusion-transmitted infections should be reduced by stringent screening of donors, restrictive transfusion practice and certain interventions like irradiation of blood units before transfusion. A recent international survey of transfusion practices for extremely premature infants showed that factors considered “very important” regarding the need to administer blood transfusions included degree of oxygen requirement and need for respiratory support. Donor exposure can also be reduced by allocating single donor units, split into paedipacks, to babies predicted to need more than one transfusion episode within the expiry date of the donation. Implementing a program to improve compliance with neonatal intensive care unit transfusion guidelines was accompanied by a reduction in transfusion rate. The implementation of guidelines to improve compliance with neonatal intensive care unit transfusion guidelines can also reduce the transfusion rate.

Advancements in Cortisol Detection: From Conventional Methods to Next-Generation Technologies for Enhanced Hormone Monitoring.

The hormone cortisol, released as the end-product of the hypothalamic-pituitary-adrenal (HPA) axis, has a well-characterized circadian rhythm that enables an allostatic response to external stressors. When the pattern of secretion is disrupted, cortisol levels are chronically elevated, contributing to diseases such as heart attacks, strokes, mental health disorders, and diabetes. The diagnosis of chronic stress and stress related disorders depends upon accurate measurement of cortisol levels; currently, it is quantified using mass spectroscopy or immunoassay, in specialized laboratories with trained personnel. However, these methods are time-consuming, expensive and are unable to capture the dynamic biorhythm of the hormone. This critical review traces the path of cortisol detection from traditional laboratory-based methods to decentralised cortisol monitoring biosensors. A complete picture of cortisol biology and pathophysiology is provided, and the importance of precision medicine style monitoring of cortisol is highlighted. Antibody-based immunoassays still dominate the pipeline of development of point-of-care biosensors; new capture molecules such as aptamers and molecularly imprinted polymers (MIPs) combined with technologies such as microfluidics, wearable electronics, and quantum dots offer improvements to limit of detection (LoD), specificity, and a shift toward rapid or continuous measurements. While a variety of different sensors and devices have been proposed, there still exists a need to produce quantitative tests for cortisol ─ using either rapid or continuous monitoring devices that can enable a personalized medicine approach to stress management. This can be addressed by synergistic combinations of technologies that can leverage low sample volumes, relevant limit of detection and rapid testing time, to better account for cortisol's shifting biorhythm. Trends in cortisol diagnostics toward rapid and continuous monitoring of hormones are highlighted, along with insights into choice of sample matrix.

Stem cell-derived vessels-on-chip for cardiovascular disease modeling

The metabolic syndrome is accompanied by vascular complications. Human invitro disease models are hence required to better understand vascular dysfunctions and guide clinical therapies. Here, we engineered an open microfluidic vessel-on-chip platform that integrates human pluripotent stem cell-derived endothelial cells (SC-ECs). The open microfluidic design enables seamless integration with state-of-the-art analytical technologies, including single-cell RNA sequencing, proteomics by mass spectrometry, and high-resolution imaging. Beyond previous systems, we report SC-EC maturation by means of barrier formation, arterial toning, and high nitric oxide synthesis levels under gravity-driven flow. Functionally, we corroborate the hallmarks of early-onset atherosclerosis with low sample volumes and cell numbers under flow conditions by determining proteome and secretome changes in SC-ECs stimulated with oxidized low-density lipoprotein and free fatty acids. More broadly, our organ-on-chip platform enables the modeling of patient-specific human endothelial tissue and has the potential to become a general tool for animal-free vascular research.

A simple, fast and green method for API gravity, density, sulfur and nitrogen determination in crude oil by ATR-FTIR

In this work, the combination of multivariate data analysis and Fourier-transformed infrared (FTIR) for the characterization of crude oil samples based on the determination of API gravity (°API), density, sulfur and nitrogen contents is proposed. Reference values for density and °API were determined according to ASTM D7042-04, while nitrogen was determined according to ASTM D4629. For sulfur determination, a miniaturized MAD-SRC method was used to obtain reference values. The best model for density and °API was obtained applying a partial least squares with two response variables (PLS2) calibration. Multiplicative scattering correction (MSC) and mean centering pre-treatments were applied for this model (R2 of 0.936 and 0.944 for density and °API, respectively). For N content, the best PLS model consisted in using selected variables and first derivative with Savitsky-Golay smoothing (R2 = 0.983). The PLS model for S content prediction was built using selected variables and applying first derivative with Savitsky-Golay smoothing and mean-centering data pre-treatment (R2 = 0.982). The environmental impact and greenness of crude oil characterization applying both the alternative (using multivariate calibration) and the official methods was evaluated using the White Analytical Chemistry (WAC) tool. WAC analysis resulted in relatively low scores for conventional methods (76.8 for ASTM D4629, 75.8 for ASTM D7042-04 and 70.7 for MAD-SRC) while the ATR-FTIR method was considered the greener method (low impact, WAC score of 98.3). The proposed method combining FTIR with multivariate calibration enabled the use of a very low sample volume (about 0.01 mL), minimum waste generation, and suitable results were achieved, saving time and reagents.

VitroJet: new features and case studies.

Single-particle cryo-electron microscopy has become a widely adopted method in structural biology due to many recent technological advances in microscopes, detectors and image processing. Before being able to inspect a biological sample in an electron microscope, it needs to be deposited in a thin layer on a grid and rapidly frozen. The VitroJet was designed with this aim, as well as avoiding the delicate manual handling and transfer steps that occur during the conventional grid-preparation process. Since its creation, numerous technical developments have resulted in a device that is now widely utilized in multiple laboratories worldwide. It features plasma treatment, low-volume sample deposition through pin printing, optical ice-thickness measurement and cryofixation of pre-clipped Autogrids through jet vitrification. This paper presents recent technical improvements to the VitroJet and the benefits that it brings to the cryo-EM workflow. A wide variety of applications are shown: membrane proteins, nucleosomes, fatty-acid synthase, Tobacco mosaic virus, lipid nanoparticles, tick-borne encephalitis viruses and bacteriophages. These case studies illustrate the advancement of the VitroJet into an instrument that enables accurate control and reproducibility, demonstrating its suitability for time-efficient cryo-EM structure determination.

Label-Free detection of Poly-Cystic Ovarian Syndrome using a highly conductive 2-D rGO/MoS2/PANI nanocomposite based immunosensor

Polycystic ovarian syndrome (PCOS) is an endocrinal disorder characterized by multiple tiny cysts, amenorrhea, dysmenorrhea, hirsutism, and infertility. The current diagnostic tools comprise of expensive, time-consuming ultrasonography to serological test, which have low patient compliance. To address these limitations, we have developed a highly sensitive, cost effective and ultrafast immunosensor for the diagnosis of PCOS. Herein, we have fabricated a 2-D electro conductive composites of reduced Graphene oxide (rGO), Molybdenum disulfide (MoS2), and Polyaniline (PANI) as electrode material. Furthermore, for detecting an early and non-cyclic biomarker of PCOS, i.e. anti-Mullerian hormone (AMH). We utilize the specific antigen–antibody mechanism, in which monoclonal Anti-AMH antibodies were covalently immobilized using EDC-NHS chemistry on electrode. The developed biosensor was physicochemical and electrochemically characterized to demonstrate its efficiency. Further we have investigated the biosensor’s performance with Cyclic Voltammetry, Differential Pulse Voltammetry, and Electrochemical Impedance Spectroscopy. We have validated that under the optimized condition the immunosensor exhibits higher sensitivity with a LOD of ∼ 2.0 ng/mL with a linear range up to 100 ng/mL. Furthermore, this immunosensor works efficiently with a lower sample volume (>5 μL), which provides a sensitive, reproducible, low-cost, rapid analysis to detect AMH level in PCOS diagnosis.

Smartphone-Based Colorimetric Microfluidic Paper-Based Analytical Device for On-Site Detection of Calcium Ions in Milk Samples

Milk is a major dietary source of calcium; thus, accurate and precise quantification of calcium in milk and other dairy products is necessary for quality control. Conventional instrumental methods are sensitive and accurate but require expensive instrumentation, sample pretreatment, skilled personnel, and a long detection time. Herein, a microfluidic paper-based analytical device (µPAD) was developed to quantify calcium ions (Ca2+) in milk using a smartphone as a detector. The method utilized a complexometric reaction between Ca2+ and ethylene diamine tetraacetic acid with murexide as an indicator. The response was the average color intensity at the detection zones measured using the Color Picker application. The proposed µPAD exhibited a good linear detection range (1 to 10 mM), a low detection limit of 0.42 mM, and a short analysis time of 2 min. The determination of Ca2+ in milk samples using the proposed µPAD was consistent with a potentiometric method. The proposed µPAD demonstrates many advantages, including rapid detection, acceptable accuracy, low cost, low sample volume, and suitability for on-site quantification of Ca2+ in real samples.

Bio-Interface Analysis and Detection of Aβ using GaN HEMT-based Biosensor

Early detection, prognosis, and diagnosis of devastating neurological disorders such as TBI and AD are crucial for developing treatment strategies, efficient patient outcomes, and management in biomedical fields. The work reports the design, development, suitability analysis, and validation of a label-free GaN HEMT-based biosensing platform for the non-invasive detection of FDA-approved biomarker Aβ in saliva sample concentration ranges. The biofunctionalization assay has been validated with both electrical and FTIR spectroscopy-based measurements. A comparative analysis with ELISA assay shows good agreement with ∼2.0% measurement errors demonstrating platform stability and accuracy for Aβ detection. The platform offers a peak sensitivity of 27.20 μA/pg ml−1, 19.10 μA pg−1 ml−1, and 1.48 μA/pg ml−1 for detection in saliva, serum, and CSF concentration ranges with high specificity, excellent repeatability, and reproducibility of the results. The platform offers a sensitivity of 2.40 μA/pg ml−1, 15.40 μA pg−1 ml−1, and 27.20 μA/pg ml−1 for Sbias of 1.0 V, 3.3 V, and 5.0 V respectively. The key biosensor features include fast detection with a response time of 5–10 s and a low sample volume requirement of ∼1–2 μl. The platform comparison with ELISA shows a similar and acceptable linearity trend. A novel equation has been established for ELISA and developed platform-based detection for possible detection accuracy and validation useful for correlating the sensor response with ELISA test results and vice-versa for any target Aβ concentrations. To the best of our knowledge, this is the first time reporting of Aβ detection using a GaN HEMT-based biosensing platform.

Network Screening on Low-Volume Roads Using Risk Factors

This paper proposes a new method for network screening on rural low-volume roads. These roads are important as they provide critical access to agricultural land and tourist attractions. Most low-volume roads belong to the lowest functional class (local rural roads) and thus are built to lower design standards. Conventional hot spot network screening techniques may not be appropriate for low-volume roads due to the sporadic nature of crashes occurring on these roads. Conversely, sophisticated network screening approaches require extensive roadway and traffic data that are often unavailable to local agencies due to a lack of resources, and/or a lack of technical expertise. This research attempts to address these obstacles to low-volume road network screening which aims to identify candidate sites for safety improvements. The research used an extensive low-volume road sample from the state of Oregon and Empirical Bayes expected number of crashes in developing the proposed models for network screening. The proposed models do not require exact measurements of roadway geometric features as all geometric variables were classified into categories that are easy to compile by local agencies. Further, the method could be used with and without traffic data, without compromising the effectiveness of the network screening process.

One-Step Detection of Vancomycin in Whole Blood Using the Lateral Flow Immunoassay.

Vancomycin (VAN) is an effective antibiotic against Gram-positive bacteria and the first-line therapy to prevent and treat methicillin-resistant Staphylococcus aureus (MRSA) and severe infections. However, low concentrations of VAN can result in resistant strains. High doses of VAN can cause nephrotoxicity and ototoxicity; thus, VAN is a representative drug for which drug monitoring is recommended. Several methods have been proposed to detect VAN. Among them, lateral flow immunoassays (LFIAs) have advantages, such as simple and user-friendly operation, low sample volume requirement, and cost effectiveness. In this study, we developed an LFIA capable of rapid on-site detection such that the VAN concentration in plasma could be monitored within 20 min by a one-step detection process using whole blood without plasma separation. VAN can be detected in whole blood over a wide range of concentrations (20-10,000 ng/mL), and the LFIA reported here has a detection limit of 18 ng/mL. The applicability of the developed LFIA compared to the results of measuring VAN with a commercial enzyme-linked immunosorbent assay kit showed a satisfactory correlation (Spearman's rho, ρ = 0.891). Therefore, the developed LFIA enables rapid and wide-range VAN detection in whole blood and can aid in drug monitoring to evaluate patients' responses to treatment.

Real-time continuous gesture recognition system based on PSO-PNN

In the field of natural human–machine interaction, wearable gesture interaction technologies have received considerable attention, particularly continuous gesture (CG) recognition. However, CG faces several challenges, including the impact of motion characteristics on gesture recognition and performance that is not sufficiently robust. Traditional algorithms are highly dependent on samples, thus meeting the requirements of low sample volume and high accuracy simultaneously is challenging. To address these challenges, we propose a real-time CG recognition system based on particle swarm optimization (PSO) and probabilistic neural network (PNN). This system employs principal component analysis for signal dimensionality reduction to alleviate computational burden and uses K-means clustering and Pearson correlation coefficient to extract optimal features for gesture classification. In offline gesture recognition experiments involving six CGs, the algorithm achieved an accuracy rate of 97% with a training set of 300 samples and a runtime of just 31.25 ms. Compared to other five algorithms, the proposed algorithm improved accuracy by at least 9% and reduced the runtime by 40.475 ms. Moreover, gesture recognition experiments were conducted using different datasets, with the PSO-PNN algorithm achieving an average recognition rate of 90.17%, at least 9.84% higher than other algorithms. Finally, in experiments on online CG control for robots in complex environments, the PSO-PNN demonstrated real-time performance of 28.56 ms and a task completion rate of 90.67%, validating the feasibility of PSO-PNN. This research provides a substantial theoretical and technical foundation for the ongoing enhancement and application of CG recognition technology.

Polyacrylamide degradation in oil field wastewater by UV/H2O2 and UV/PDS: Rapid experimental measurement and model simulation

Ultraviolet-based advanced oxidation processes (UV-AOPs) are effective for degrading refractory pollutants in wastewaters. However, performing rapid efficiency evaluations represents a significant challenge for process selection and optimization in practical application. This study investigated polyacrylamide (PAM) degradation by UV/hydrogen peroxide (H2O2) and UV/potassium peroxydisulfate (PDS) in pure water and practical oil field wastewater. Based on a previously developed mini-fluidic photoreaction system that is easy to operate and requires low sample volumes, an experimental setup for rapid evaluation of UV-AOP efficiency was constructed that enabled on-line analyses of viscosity, UV absorbance, and pH via specially fabricated flow cell devices together with off-line analyses of PAM, chemical oxygen demand (COD), and acrylamide (AM). The PAM degradation rate constants (k′PAM) in practical oil field wastewater by UV/H2O2 and UV/PDS were 2.4 and 2.1 m2 einstein−1, respectively, which were much higher than those by sole UV process (0.8 m2 einstein−1). Combing with the analyses of viscosity, UV absorbance (220 nm), pH, COD, and AM concentration, the UV/H2O2 and UV/PDS demonstrated the effectiveness in PAM degradation in oil field wastewater. Additionally, the scavenging capacities of HO• and SO4•− of practical oil field wastewater were determined, and the k′PAM values were predicted by model simulation and verified experimentally. This study provides rapid evaluation methods for UV-AOP efficiencies, which is helpful for selecting and optimizing UV-AOPs for industrial wastewater treatments.

Detecting thyrotropin receptor mRNA from peripheral blood of patients with differentiated thyroid cancer rules out non-aggressive cases.

Early diagnosis of thyroid cancer is hampered by the inability of fine-needle aspiration biopsy (FNAB) to accurately classify ∼30% of cases while preoperative cancer staging detects lymph nodal involvement in only half of cases. Liquid biopsy may present an accurate, non-invasive alternative for preoperative thyroid nodule assessment. Thyrotropin receptor (TSHR) mRNA, a surrogate marker for circulating cancer cells (CTC), may be an option for early detection of malignancy from peripheral blood, but requires methodological improvements. We aimed to investigate if TSHR mRNA can be detected in low sample volumes by employing an ultrasensitive method - droplet digital PCR (ddPCR). Less than 5mL of blood was collected from 47 patients with thyroid nodules (25 benign and 22 malignant). RNA was isolated from the fraction of mononuclear cells where CTCs segregate. Samples were analysed for the presence of TSHR mRNA by ddPCR. Thyrotropin receptor mRNA was detectable in 4mL sample volumes, with the test having good specificity (80%) but modest diagnostic accuracy (68.1%). Combining TSHR mRNA with ultrasound features and FNAB diagnosis, the test reaches high rule-out performances (sensitivity = 90% and NPV = 88.2%). Strikingly, TSHR mRNA correctly classified all samples with thyroid capsule invasion, lymph node metastasis and extrathyroidal extension. If aggressiveness is defined using these parameters, TSHR mRNA test reaches 100% sensitivity and 100% NPV for detecting high-risk cases. Employing ddPCR for TSHR mRNA improves its measurement by enabling detection in sample volumes common for laboratory testing. The test displays high prognostic performance, showing potential in preoperative risk assessment.

Methods for molecular characterization of dissolved organic matter in the alpine water environment: an overview

The alpine area has become a sensitive indicator and amplifier of global climate change and human activities because of its unique geographical and climatic conditions. Being an essential part of biochemical cycling, dissolved organic matter (DOM) could effectively help understand the process, structure, and function of alpine aquatic ecosystems. Due to the low content and sampling difficulties, the analysis of DOM in alpine water demands high sensitivity with low sample volume, which has not been comprehensively reviewed. This review summarizes the DOM sampling, pretreatment, and analysis methods involving the characterization of concentration, spectroscopy, and molecular structure. Overall, conventional parameters are the basis of advanced characterization methods. Spectroscopic tests can reveal the optical properties of DOM in response to lights from ultraviolet to infrared wavelengths, to distinguish the chemical composition. Molecular structure characterizations can provide microscopic information such as functional groups, element ratios, and molecular weights. The combination of multiple methods can depict DOM composition from multiple perspectives. In sum, optimized sampling and pretreatment, high-sensitivity molecular characterization, and method integration are crucial for effectively analyzing DOM components in alpine waters. These perspectives help to standardize the DOM characterization process and to understand the correlation between DOM composition and its properties, as well as the migration and transformation of DOM.

Validation of a plasmonic-based serology biosensor for veterinary diagnosis of COVID-19 in domestic animals

The coronavirus disease 2019 (COVID-19) pandemic recently demonstrated the devastating impact on public health, economy, and social development of zoonotic infectious diseases, whereby viruses jump from animals to infect humans. Due to this potential of viruses to cross the species barrier, the surveillance of infectious pathogens circulation in domestic and close-to-human animals is indispensable, as they could be potential reservoirs. Optical biosensors, mainly those based on Surface Plasmon Resonance (SPR), have widely demonstrated its ability for providing direct, label-free, and quantitative bioanalysis with excellent sensitivity and reliability. This biosensor technology can provide a powerful tool to the veterinary field, potentially being helpful for the monitoring of the infection spread. We have implemented a multi-target COVID-19 serology plasmonic biosensor for the rapid testing and screening of common European domestic animals. The multi-target serological biosensor assay enables the detection of total SARS-CoV-2 antibodies (IgG + IgM) generated towards both S and N viral antigens. The analysis is performed in less than 15 min with a low-volume serum sample (<20 μL, 1:10 dilution), reaching a limit of detection of 49.6 ng mL−1. A complete validation has been carried out with hamster, dog, and cat sera samples (N = 75, including 37 COVID-19-positive and 38 negative samples). The biosensor exhibits an excellent diagnostic sensitivity (100 %) and good specificity (71.4 %) for future application in veterinary settings. Furthermore, the biosensor technology is integrated into a compact, portable, and user-friendly device, well-suited for point-of-care testing. This study positions our plasmonic biosensor as an alternative and reliable diagnostic tool for COVID-19 serology in animal samples, expanding the applicability of plasmonic technologies for decentralized analysis in veterinary healthcare and animal research.

Application of a bar adsorptive microextraction based methodology for doping control of alkylamine stimulants in urine matrices

To comply with ‘World Anti-Doping Agency’ (WADA) guidelines, doping control laboratories must continuously adjust their analytical procedures. Therefore, sample preparation continues to play a critical step in modern analytical strategies, namely by replacing the tedious, time and solvent consuming commonly employed (e.g. liquid-liquid extraction). The present contribution proposes, for the first time in doping control, bar adsorptive microextraction (BAμE) as an alternative analytical technique for the qualitative determination of six alkyl amine stimulants (AAs; 1,3-dimethylbutylamine, 1,4-dimethylpentylamine, heptaminol, isometheptene, octodrine and tuaminoheptane) in urine matrices followed by derivatization prior to gas chromatography coupled to mass spectrometry, operating in the selected ion monitoring mode acquisition (GC–MS(SIM)). After selecting the most selective coating phase, i.e., a mixed-mode reversed phase/strong anion exchange sorbent (P2), assays performed under optimized experimental conditions [microextraction - BAμE(P2), 1 h (1,000 rpm), pH 11, 10 % NaCl; back-extraction - methanol (150 μL), 30 min, under sonication], allowed remarkable recoveries ranging from 48.7 % (heptaminol, 200 ng/mL) to 83.1 % (1,4-dimethylpentylamine, 200 ng/mL). The validation assessment assays of the proposed methodology showed suitable limits of identification (5.0–35.0 ng/mL), appropriate linear dynamic ranges (5.0–200.0 ng/mL) and good determination coefficients (r2 > 0.9937), as well as excellent selectivity, robustness, accuracy and precision. To check whether the methodology is fit-for-purpose, four previously analysed proficiency urine samples were successfully tested, in which were unequivocally detected and identified some of the target AAs. The present methodology showed to be a remarkable alternative in comparison to other dedicated analytical approaches to screen AAs in urine matrices, since it is cost-effective, user- and eco-friendly, requiring low volume of urine sample (1 mL). The great potential of this analytical technology in doping control lies in a very effective microextraction combined with the minimization of potential interferents, presenting itself as an added value to be applied to other types of substances and complex matrices.

Biocatalytic ZIF-8 surface-functionalized micromotors navigating in the cerebrospinal fluid: toward Alzheimer management.

Alzheimer's disease (AD) is the major cause of irreversible dementia in the elderly population worldwide and one of the major causes of the decrease in the quality of life. Efficient diagnosis and monitoring would allow a fast treatment to delay the appearance of symptoms. Herein, zeolitic imidazole framework (ZIF-8)@Au@catalase micromotors are described for motion-based sensing of copper as a marker of AD. The synthesis design was based on enzyme covalent immobilization instead of encapsulation to maximize the contact with the sample at the microscale for the potential use of extremely low AD-diagnosed sample volumes. The micromotors are prepared by asymmetric modification of ZIF-8 with a gold layer for functionalization of catalase as a compatible biocatalyst. The micromotors can propel at speeds of up to 287 ± 41 μm s-1 in cerebrospinal fluid (CSF) samples of healthy volunteers. Yet, in the presence of copper, catalase poisoning results in a decrease in the speed that can be monitored for motion-based sensing detection, as illustrated in the analysis of CSF samples from AD patients from mild to severe stages (Braak III to Braak VI). The copper-mediated modulation of catalase activity proposed here as an indicator of progression states in AD disease possesses distinct advantages such as ultrafast analysis (less than 1 min) and requiring only 1 μL of sample, holding considerable promise as a supporting prescreening tool for fast diagnosis of AD and other neurodegenerative diseases.

Current trends in electrochemical approaches for liver biomarker detection: a mini-review.

Aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin, gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), and albumin are well-established liver biomarkers with significant physiological functions. Alterations in these liver function tests can be indicative of the presence and progression of acute and chronic liver conditions such as liver cirrhosis, non-alcoholic fatty liver disease, biliary disease, and liver failure. Therefore, accurate and quantitative detection of these biomarkers is crucial for diagnosing and monitoring liver disease. There are several commercially available chemistry analyzers capable of simultaneously detecting all these biomarkers, as well as numerous biosensors designed for individual detection. Various techniques have been employed, including colorimetry, surface-enhanced Raman spectroscopy (SERS), electrochemiluminescence (ECL), fluorescence-based techniques, and electrochemical methods. Among these, electrochemical detection stands out due to its simplicity, cost-effectiveness, low sample volume requirement, label-free detection, high sensitivity, fast response times, miniaturization, and portability. Information on recently developed electrochemical biosensors is summarized through detailed tables and is intended to guide future research and development efforts in this area.