Ml Of Sample Research Articles

Seasonally Dependent Daytime and Nighttime Formation of Oxalic Acid Vapor and Particulate Oxalate in Tropical Coastal and Marine Atmospheres

Oxalic acid is the most abundant low-molecular-weight dicarboxylic acid in the atmosphere, and it plays a crucial role in the formation of new particles and cloud condensation nuclei. However, most observational studies have focused on particulate oxalate, leaving a significant knowledge gap on oxalic acid vapor. This study investigated the concentrations and formation of oxalic acid vapor and oxalate in PM2.5 at a rural tropical coastal island site in south China across different seasons, based on semi-continuous measurements using an Ambient Ion Monitor-Ion Chromatograph (AIM-IC) system. We replaced the default 25 μL sampling loop on the AIM-IC with a 250 μL loop, improving the ability to distinguish the signal of oxalic acid vapor from noise. The data revealed clear seasonal patterns in the dependent daytime and nighttime formation of oxalic acid vapor, benefiting from high signal-to-noise ratios. Specifically, concentrations were 0.059 ± 0.15 μg m−3 in February and April 2023, exhibiting consistent diurnal variations similar to those of O3, likely driven by photochemical reactions. These values decreased to 0.021 ± 0.07 μg m−3 in November and December 2023, with higher nighttime concentrations likely related to dark chemistry processes, amplified by accumulation due to low mixing layer height. The concentrations of oxalate in PM2.5 were comparable to those of oxalic acid vapor, but exhibited (3–7)-day variations, superimposed on diurnal fluctuations to varying degrees. Additionally, thermodynamic equilibrium calculations were performed on the coastal data, and independent size distributions of particulate oxalate in the upwind marine atmosphere were analyzed to support the findings.

Conjugation of Short-Chain Fatty Acids to Bicyclic-Amines for Analysis by Liquid Chromatography Tandem Mass Spectroscopy.

Conjugation of short-chain fatty acids (SDFAs) to amines containing ring structures allows for better measurement by liquid chromatography tandem mass spectroscopy (LC-MS/MS). However, collision-induced dissociation (CID) results in breaking the conjugate back to the original SCFA and amine. We therefore set out to find an amine that would remain on the SCFA after CID and create a unique daughter for selectivity of measurement. Of twenty-seven amines with ring structures, we found four that contain bicycle-type structures (two rings connected by a carbon) with nitrogen in the second ring. CID removes the second ring at the nitrogen, leaving the first ring on the daughter. Of the four amines, 4-(pyrrolidine-1-ylmethyl) benzylamine (4PyBA) showed the strongest conjugation. Conjugation of 4PyBA to SCFA (C3-C6), their isomers and their phenylated versions (and isomers) resulted in good chromatographic peaks and separation. CID resulted in unique daughters that allowed for selectivity of measurement. Using this method, standard curves were generated that show good linearity (r2 > 0.99) in the nM and μM range with lower limits of detection between 40 and 229 nM for a 10 μL sample. Finally, we used this method to measure SCFA in plasma, liver, platelets, and red blood cells, demonstrating its use in biological systems. Because SCFAs are an index of microbiome diversity in the gastrointestinal track, this method will allow us to study changes in SCFAs and the microbiome in pathologic conditions including trauma, hemorrhage, and sepsis.

Epididymal bull sperm selection by Percoll® density-gradient centrifugation prior to conventional or ultra-rapid freezing enhances post-thaw sperm quality.

This study evaluated the effectiveness of Percoll® density gradient centrifugation (Percoll-DGC) for selecting bull epididymal sperm prior to conventional slow (CS) or ultra-rapid (UR) freezing and its effects on sperm quality. Fifteen pooled samples from 30 epididymides (2 different samples/pool) of 15 bulls were split into two aliquots assigned to either CS or UR freezing. Samples were either selected using Percoll-DGC (40/80 %) or left non-selected (control), resulting in four pre-freezing treatments: Percoll-CS, Control-CS, Percoll-UR, and Control-UR. The CS freezing used 5% glycerol, exposing sperm straws to liquid nitrogen (LN2) vapors, while UR freezing used 100 mM sucrose with direct submersion of 30 μL samples into LN2. Overall, sperm quality was higher in CS treatments than in UR treatments. Pre-freezing, Percoll-CS improved straight-line velocity (VSL), linearity (LIN), and beat-cross frequency (BCF) compared to Control-CS (P < 0.05). Similarly, Percoll-UR treatment enhanced progressive motility (PSM), velocities, straightness (STR), amplitude of lateral head displacement (ALH), and BCF compared to Control-UR (P < 0.05). Post-thaw, Percoll-CS demonstrated higher kinematic parameters, viability, and acrosome integrity compared to Control-CS (P < 0.05). Meanwhile, Percoll-UR improved viability and acrosome integrity relative to Control-UR (P < 0.05). Notably, both Percoll-UR and Control-UR resulted in lower DNA fragmentation compared to Percoll-CS. In conclusion, Percoll-DGC selection prior to CS freezing significantly improved post-thaw sperm quality, including kinematics, viability, and acrosome integrity. For UR freezing, Percoll-DGC primarily enhanced post-thaw viability and acrosome integrity.

An automated blood test for glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) to predict the absence of intracranial lesions on head CT in adult patients with mild traumatic brain injury: BRAINI, a multicentre observational study in Europe

Following mild traumatic brain injury (mTBI), elevated concentrations of brain-specific blood proteins glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase-L1 (UCH-L1) may be indicative of intracranial lesions normally detected by head CT scans. We sought to validate the performance of this combination of biomarkers at predetermined cutoff values with an automated immunoassay to predict which patients did not have intracranial lesions. This prospective, observational study was conducted in France and Spain at 16 emergency departments. Adult patients with mTBI were eligible if they had a head CT scan and gave a 10-ml blood sample within 12h of injury. GFAP and UCH-L1 serum concentrations were measured and analysed, in less than an hour time, according to predefined cutoff values of 22pg/ml and 327pg/ml, respectively. Serum concentrations of S100B protein were concomitantly determined in a subset of patients. The primary outcome measures were the sensitivity and negative predictive value (NPV) of the combined GFAP-UCH-L1 test to rule out intracranial lesions on head CT scans. gov (NCT04032509). Between August 2019 and June 2021, 1508 patients were recruited, and 1438 were included in the main analysis. Median age was 69 years (IQR 44-83). Most patients (74%) presented 3h after trauma. 179 (12.4%) patients were positive for intracranial lesions by CT. The sensitivity of the combined test was 98.3% (95% CI 95.0-99.7) and the specificity 24.9 (95% CI 22.6-27.4), with a NPV of 99.1% (95% CI 97.1-99.8). Three patients with a positive CT scan had negative biomarker test results. S100B had a sensitivity of 83.0% (95% CI 76.2-88.2) and a NPV of 94.2% (95% CI 91.6-96.0). Patients with higher biomarker values more frequently had poorer recovery at 3 months after injury. Testing for GFAP and UCH-L1, using validated cutoffs obtained with a new, fast automated immunoassay platform, accurately predicted the absence of intracranial lesions on head CT following mTBI. This study is co-funded by the European Institute of Innovation and Technology (EIT) Health, a body of the European Union (Grant nº19474). Biomarkers tests were funded by bioMérieux.

Tetrahedral DNA nanostructures-assisted electrochemical assay for detecting circulating tumor DNA by combining a masking tactic with 3D-hybridization chain reactions

Circulating tumor DNA (ctDNA) is a remarkable noninvasive tumor marker that plays a crucial role in tumor diagnosis, prognosis and treatment. However, detecting low-abundance ctDNA from a substantial amount of nucleic acids originating from healthy cells is challenging. Herein, we proposed a tetrahedral DNA nanostructures (TDNs)-assisted electrochemical biosensor for ctDNA detection. This biosensor combines a masking tactic with 3D-hybridization chain reactions. Masking hairpins (MHs) were initially introduced to prevent interference from wild-type (WT) DNA. Then, the initiator sequence was transferred to the electrode surface modified with TDNs by the target ctDNA. The initiator sequence triggers the 3D self-assembly of hairpin strands, leading to the formation of DNA networks or even DNA hydrogels (long reaction time). This process generates numerous evenly distributed biotin molecules that can bind to streptavidin peroxidase to considerably amplify the signal. This method exhibits high sensitivity (the minimum concentration for detecting ctDNA is 1 aM, which corresponds to 60 ctDNA molecules in 100 μl sample) and excellent specificity (single mismatch). More importantly, this high-performance sensor can detect ctDNA with other mutation sites and their mixtures by modifying the corresponding capture probes on the TDNs. Furthermore, this ultrasensitive sensor effectively detects target ctDNA (0.001%) at high levels of WT DNA and in complex matrices such as serum. These findings suggest that the sensor has promising potential as a noninvasive tool for early tumor diagnosis.

Recyclable Cardiac Biosensor Based on Light-Triggered Antibody Regeneration Method

Introduction: The diagnostic utility of heart failure biomarkers, particularly Brain Natriuretic Peptide (BNP), is well-documented. Yet, the clinical translation of BNP for tracking the progression of heart failure and assessing the therapeutic efficacy of drugs requires advancements in wearable sensor technology. Traditional antigen-antibody reaction-based biosensors, while effective, are hampered by the need for frequent reagent replenishment due to antibody regeneration challenges. We introduce a novel method employing laser irradiation to modulate pH levels, facilitating antibody regeneration and enabling the development of a wearable BNP sensor. Methodology: Our approach utilizes a photoacid (Hydroxypyrene Trisulfonic Acid or HPTS) immobilized around the capture antibody of a sensor. Irradiating the sample with visible light for seconds induces HPTS to acidify the local environment, dissociating the bound antigens and regenerating the antibodies for reuse. The biosensor design incorporates: (a) a stainless-steel mesh substrate with gold vapor-deposited on the surface (gold mesh substrate); (b) an anti-BNP antibody immobilized on the substrate via a self-assembled monolayer; (c) HPTS immobilized on the gold mesh substrate; (d) a chamber containing the gold mesh substrate for immunoreaction; (e) a photomultiplier tube for signal detection. We validated this methodology by measuring pH changes in a 1 mL HPTS solution irradiated with a 50 mW, 405 nm laser for 5 minutes. Subsequently, a calibration curve for the BNP immunosensor was established using BNP standards (0.1 - 100 pg/mL). Finally, the light-triggered regeneration method was employed to dissociate BNP from the anti-BNP antibody. Results: Laser irradiation triggered a decrease in HPTS solution pH, which rapidly returned to its baseline upon cessation of irradiation. This phenomenon was repeatable up to three times. The sensor captured BNP from a 50 µL sample via anti-BNP antibody interaction. Subsequent introduction of enzyme-labeled detection antibody formed a BNP sandwich complex. Luminescent substrate addition allowed for luminescence intensity (I) measurement at 450 nm. The resulting calibration curve fit the equation: I = 2980 ln (BNP) + 19713 (R2 = 0.86). The gold mesh substrate enabled visible light to penetrate the reaction area, facilitating detection by the photomultiplier tube. Finally, laser irradiation in the presence of 5 mM HPTS dissociated the antigen (BNP). Conclusion: We present a novel laser-based method for antibody regeneration in biosensors, facilitating the realization of wearable devices for continuous heart failure monitoring. Overcoming the limitations of traditional biosensor reagent replenishment, our technology paves the way for advanced heart failure management tools, potentially transforming diagnostic and therapeutic strategies in cardiovascular medicine. Figure 1

Single-Molecule Detection of Serum MicroRNAs for Medulloblastoma with Biphasic Sandwich Hybridization-Assisted Plasmonic Resonant Scattering Imaging.

MicroRNA (miRNA) dysregulation is closely related to the occurrence and progression of medulloblastoma (MB). However, the full potential of serum circulating miRNAs in MB diagnosis is restricted by their ultralow abundance in peripheral blood due to blood-brain barrier. Here, we report the direct preamplification-free detection of aberrant expression of oncogenic miRNAs in serum from MB patients by proposing a simple yet robust single-molecule assay that combines biphasic sandwich hybridization in nucleic acids and the dark-field single-particle plasmonic imaging (B2S2PI). In this strategy, signal DNA was prehybridized with target miRNA in homogeneous solution to form sDNA-RNA complexes. Then the captured DNA strands with rationally adjusted surface densities could efficiently capture the sDNA-RNA complexes to generate a well-separated DNA-RNA sandwich structure. The combination of homogeneous and heterogeneous reactions enabled interface-mediated hybridization reactions to maintain molecular stability with fewer bases, making it suitable for the direct amplification-free assays of short miRNA targets. Labeling the DNA-RNA hybrids with plasmatic gold nanotags allowed nondestructive recognition and imaging of individual miRNA targets under mild conditions with high signal-to-noise ratio. By digitally counting and analyzing the bright plasmonic resonant scattering spots, B2S2PI enabled both the measurement of a low femtomolar concentration of circulating miRNA-21 in 5 μL sample volume within a turnaround of 2 h and the discrimination of single base mismatches. Moreover, B2S2PI was universal for detecting miRNAs with different sequences and secondary structures. Further analysis of clinical serum samples revealed that B2S2PI was capable of accurately distinguishing MB patients from noncancer controls with an area under the curve (AUC) of 0.99, which was superior to that of qRT-PCR. B2S2PI holds promise as a novel alternative means for single-molecule miRNA assay and sheds light on the circulating nucleic acid-based liquid biopsy of intracranial malignant tumors.

CRISPR-Cas14a and allosteric transcription factors empowered cell-free electrochemical biosensor for highly sensitive and stable detection of progesterone in multiple scenarios

In this study, a cell-free electrochemical assay based on allosteric transcription factors (aTFs) and CRISPR-Cas14a was developed for the detection of progesterone in trace samples. This electrochemical biosensor helps to overcome the drawbacks of the traditional fluorescence assay based on the CRISPR-Cas system and aTFs combined for non-nucleic acid targets that is poorly effective for the detection of colored samples. By comparing and optimizing the concentration and length of the probes in the straight chain and hairpin structure, the sensor performance was improved. In addition, different sgRNA from other studies was designed to overcome the effect of sequence folding in the space region on Cas14a activation. Based on these optimization results, we constructed an electrochemical sensor for progesterone quantification in the range of 66.7pM to 3.33 × 10−1μM. This method requires only 2 μL of sample and does not necessitate complex pretreatment steps, with detection completed within 1.5 h. The method has been successfully applied to food, environmental, and biological samples, with recovery rates between 82.65% and 109%. This suggests that CRISPR and allosteric transcription factor-powered electrochemical detection methods have significant potential for use in the field of small molecule detection under various scenarios.

One-step detection of nanoplastics in aquatic environments using a portable SERS chessboard substrate

Nanoplastics present a significant hazard to both the environment and human health. However, the development of rapid and sensitive analysis techniques for nanoplastics is limited by their small size, lack of specificity, and low concentrations. In this study, a surface-enhanced Raman scattering (SERS) chessboard substrate was introduced as a multi-channel platform for the pre-concentration and detection of nanoplastics, achieved by polydomain aggregating silver nanoparticles (PASN) on a hydrophilic and a punched hydrophobic PVDF combined filter membrane. Through a straightforward suction filtration process, nanoplastics were captured by the PASN gap in a single step for subsequent SERS detection, while excess moisture was promptly eliminated from the filter membrane. The PASN-based SERS chessboard substrate, benefiting from the enhanced electromagnetic (EM) field, effectively discriminated polystyrene (PS) nanoplastics ranging in size from 30 nm to 1000 nm. Furthermore, this substrate demonstrated favorable repeatability (RSD of 8.6 %), high sensitivity with a detection limit of 0.001 mg/mL for 100 nm of PS nanoplastics, and broad linear detection ranges spanning from 0.001 to 0.5 mg/mL (R2 = 0.9916). Additionally, the SERS chessboard substrate enabled quantitative analysis of nanoplastics spiked in tap and lake water samples. Notably, the entire pre-concentration and detection procedure required only 3 μL of sample and could be completed within 1 min. With the accessibility of portable detection instruments and the ability to prepare substrates on demand, the PASN-based SERS chessboard substrate is anticipated to facilitate the establishment of a comprehensive global nanoplastics map.

B-177 Development of a Contrived Fecal Matrix for Clinical Validation of a LC-MS/MS Assay to Quantify Microbiota-derived Metabolites in Stool Specimens

Abstract Background The gut microbiota plays a critical role in health and its disruption can impact clinical outcomes in a variety of circumstances, including graft-versus-host disease following hematopoietic stem cell transplantation, Clostridioides difficile infections, and liver disease. Metagenomic and metabolomic analyses of fecal material is frequently performed in the research setting; however, evaluation of the gut microbiota is not yet incorporated into routine patient care. One barrier to its implementation is the lack of matrix-appropriate materials for validation of clinical assays. While surrogate fecal matrices are being developed for metagenomic sequencing, matrices specific for targeted metabolomics are currently unavailable. In this study, we designed a contrived fecal matrix to validate a new LC-MS/MS method for measurement of microbiota-derived metabolites from stool specimens. Methods Common food products and mucin were individually analyzed by LC-MS/MS for quantities of three microbiota-derived metabolites - butyrate, deoxycholic acid (DCA) and taurocholic acid (TCA). Food items with the lowest endogenous metabolite concentrations were selected to create a contrived fecal matrix with a consistency similar to human stool specimens. Calibrators and quality controls (QC) were then created by spiking butyrate, DCA, and TCA into either the contrived fecal matrix or 50% methanol (MeOH). The internal standards (ISTD) used were d7-butyrate, d4-DCA, and d4-TCA, respectively. Three human stool specimens were selected and spiked at a range of metabolite concentrations to assess matrix effects. All samples, calibrators and controls were homogenized in 80% MeOH extraction solvent containing the ISTDs using beadruptor tubes. The suspension was centrifuged and 25 µL of supernatant was derivatized using 12.5 µL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 12.5 µL 3-nitrophenylhydrazine at 40°C for 30 minutes. For optimal separation of isomers, 2 µL of sample was injected and separated on a CORTECS T3 column (1.6 µm, 2.1 x 100 mm, Waters) at 45°C with a flow rate of 0.350 mL/min and run-time of 20 min using the Exion LC AD (Sciex). Analytes were detected using a Sciex QTRAP 6500 mass spectrometer with an electrospray ionization source. Results A contrived fecal matrix containing a mix of avocado, black beans, gluten-free flour, and cornstarch blended in water and mucin was created. The contrived matrix was confirmed to contain minimal to no endogenous levels of the 3 metabolites of interest: butyrate, DCA, and TCA. Validation of the LC-MS/MS assay using the contrived fecal matrix showed a linear analytical measurement range from 25-2000 µM for butyrate, 1-60 µM for DCA, and 0.5-640 µM for TCA. Intra-assay precision at 7 QC levels for all analytes was &amp;lt;10%. The matrix effects from use of the contrived fecal matrix compared to patient fecal samples were 140-148% for d7-butyrate and 101-112% for d4-DCA across multiple spiked analyte concentrations. Conclusions Our LC-MS/MS method quantifies butyrate, deoxycholic acid, and taurocholic acid, metabolites which serve as indicators of microbiota health in liver disease patients. The contrived fecal matrix provides an additional tool for the development and validation of clinical assays to assess the gut microbiota.

B-155 Analysis of Monoclonal Antibodies Using a Quantification LC-MS/MS Kit - Spotlight on Infliximab

Abstract Background The use of immunoassays for measuring mAbs is well established but has some limitations, including poor performance, lack of standardization, a high cost when processing a limited number of samples, limited dynamic range, and the potential for cross-reactivity. Using LC-MS/MS associated with the ready-to-use commercial mAbXmise Kit is a simple way to implement mAbs measurement for infliximab, providing high analytical performance, ease of use, and flexibility. Methods The Promise Proteomics Inflammation mAbXmise Kit contains all the calibrators, QCs, internal standard and sample preparation consumables to run the LC-MS/MS method for infliximab. Briefly, 20µL samples are dispensed into the mAbXmise plates containing lyophilized stable labelled infliximab. Samples are transferred to the PuriXmise plate to perform the immunocapture of infliximab and allow washing of the samples. Samples are eluted into a collection plate, evaporated to dryness, resuspended, and the protease (CutXmise) is added to the sample to digest infliximab overnight. The digestion is quenched, with the samples immediately ready for analysis. Using an ACQUITY™ UPLC™ I-Class PLUS FL System, samples were injected onto a Waters™ XSelect™ Premier HSS T3 Column, eluted using a water/acetonitrile/formic acid gradient profile and quantified with both a Waters Xevo™ TQ-XS and Xevo TQ-S micro Mass Spectrometer. Results The method was shown to be linear from 2 - 100 µg/mL. Analytical sensitivity at lowest calibrator of 2µg/mL provided S/N (PtP) ≥15:1 for all tryptic peptides on both mass spectrometers. Coefficients of variation (CV) at 4 and 25µg/mL QC concentrations were all ≤ 10.0% (n = 5). The relative concentrations of the tryptic peptides were compared across 29 anonymized samples and good agreement was observed for GLE, SAV, DIL and ASA peptides. Conclusions We have demonstrated a clinical research method for quantifying Infliximab in plasma using a commercially available kit for sample preparation followed by LC-MS/MS analysis. The Promise Proteomics mAbXmise Kit aids in the day-to-day reproducibility of the immunocapture and tryptic digestion of IFX for targeted LC-MS/MS analysis with the correct peptide selection. Compared to a direct digest surrogate peptide workflow, the process used in the kit reduces matrix interference, improves analytical sensitivity, and enables the use of lower sample volumes. The method can be run on both the Xevo TQ-XS and Xevo TQ-S micro Mass Spectrometers which provide the dynamic range to quantify IFX across the expected range, and the selectivity and analytical sensitivity to obtain low-level quantification of the IFX surrogate peptide in plasma samples. The data presented combines the use of a kit dedicated to the preparation of samples and the use of liquid chromatography and mass spectrometry instrumentation to perform the quantitative analyses. The mAbXmise Kit described has not been cleared by any regulatory entity for diagnostic purposes outside of Europe. Analytical performance depends on the instrument characteristics and its settings. The end user is responsible for completion of the method development and validation. Proteomics mAbXmise Kits are not available for sale in all countries. For Research Use Only. Not for use in diagnostic procedures.

Investigating adsorption and removal of divalent Ca2+ and Pb2+ ions from aqueous solutions by gamma-irradiation using quartz tuning fork (QTF) sensor technique

In this study, gold-coated quartz tuning forks (QTFs) sensing devices functionalized with self-assembled monolayers (SAM) of a lower-rim functionalized calix[4]arene methoxy ester were used for the detection of divalent Ca2+ and Pb2+ ions in aqueous solutions by utilizing adsorption behavior and the radiative effect. The gold-coated QTF functionalized calix[4]arene methoxy ester sensing device was tested by measuring the respective frequency shifts obtained using small (60 µL) samples of aqueous PbCl2 at two different concentrations (10−6 and 10−4 M). For 10−4 M solutions of PbCl2, results showed that the resonance frequency shift Δf = 317 Hz, from 32,867 Hz (fCalix) to 32,550 Hz (fCalix⊃Pb2+) due to the absorption of lead (Pb2+) ions (10-4 M) by calixl[4]arene methoxy ester receptor molecules on the QTF sensing layer from the aqueous solution to forming the ([Calix ⊃ Pb2+]) complex. The most significant frequency changes were observed at a concentration of 10−6 M CaCl2, where CaCl2 exhibited the biggest change of 356 Hz, from 32,893 Hz (fCalix) to 32,537 Hz (fCalix⊃Ca2+), compared to 317 Hz for PbCl2 (10−4 M). The limit of detection was 100 femtomolar (fM) for CaCl2 and 245 fM for PbCl2. After that, we irradiated the receptor molecules which was holding Pb2+ ions in the complex ([calix ⊃ Pb2+]) on the QTF sensing layer with a radiation dose ranging from 7.5 to 50 µGy of gamma rays from the Cesium-137 source for 30 min. Interestingly, it was observed that the resonance frequency shift (Δf = 54 Hz) back to 32,604 Hz from 32,550 Hz (f(Calix⊃Pb2+)), which strongly suggests that the Pb2+ ion removed from ([calix ⊃ Pb2+]) complex on the QTF sensing layer due to gamma radiation dose. To follow up on the radiation effect of the ([calix ⊃ Pb2+]) on the QTF sensing layer, we stopped the gamma radiation source and kept it for an additional 10 min to see if there was any resonance frequency. It was noticed that an additional resonance frequency shifted (Δf = 33 Hz) back to 32,637 Hz from 32,604 Hz after stopping the gamma radiation source for 10 min. We assume that the complex ([calix ⊃ Pb2+]) absorbs the gamma radiation and continues the removal of Pb2+ ions from the complex on the sensing layer. A similar phenomenon was also observed for the absorption of lead (Pb2+) ions (10-6 M) by calix[4]arene methoxy ester receptor molecules on the QTF sensing layer from the aqueous solution to forming the ([Calix ⊃ Pb2+]) complex. The resonance frequency shift Δf = 142 Hz, from 32,706 Hz (fCalix) to 32,564 Hz (fCalix⊃Pb2+) due to the absorption of lead (Pb2+) ions (10-6 M) by calix[4]arene methoxy ester receptor molecules on the QTF sensing layer from the aqueous solution to forming the ([Calix ⊃ Pb2+]) complex. Subsequent exposure to gamma radiation, with doses ranging from 7.5 to 50 µGy from a Cesium-137 source over 30 min, prompted a resonance frequency shift (Δf = 37 Hz) back to 32,606 Hz from 32,564 Hz (f(Calix⊃Pb2+)). This shift strongly suggests the removal of Pb2+ ions from the ([calix ⊃ Pb2+]) complex on the QTF sensing layer due to gamma radiation dose. X-ray photoelectron spectroscopy (XPS) analysis confirmed the chemical adsorption of Pb2+ ions onto the gold-coated QTFs functionalized calix[4]arene adsorbent sensing layer surface. Therefore, the technology underlying the calix[4]arene-functionalized sensing device holds promise for diverse industrial applications, supporting potential advancements in water pollution mitigation through the proposed adsorption and irradiation mechanism.

A-215 Feasibility of a unique homogenous assay for lipoprotein(a) particle number without bias from apo(a) size isoforms

Abstract Background Lipoprotein(a) [Lp(a)] is an LDL-like particle with apolipoprotein(a) [apo(a)] covalently attached to apolipoprotein B. Elevated Lp(a) is associated with increased risk of atherosclerotic heart disease. The structural homology of Lp(a) and plasminogen and the variable number of apo(a) kringle 4 type 2 subunits (k4t2) between individuals, up to 51 repeats and a variable MW of 300-800 kDa, presents an analytical challenge. `Selection of calibrators of specific isoform size does not fix the problem, despite manufacturer’s claims to the contrary. Methods We have developed goat antisera to human apo(a)-specific peptides and a prototype immunoturbidimetric assay for Lp(a). We selected and synthesized 10 peptide sequences (15-30 aa each) distinct from the sequences found in k4t2 regions and plasminogen. Peptides were prepared attached to KLH (GenScript) and combined to immunize goats following a standard protocol including monthly boosters (Nittobo). Next, we assessed various sample volumes, reagent formulations and antisera dilutions to establish parameters on the Randox Imola analyzer. Cross-reactivity with recombinant k4t2 (40 mg/dL) and plasminogen (200 mg/dL, Athens Research) was assessed by comparison to the LOD (using delipidated plasma blank and low concentration samples). Results Titers to human Lp(a)) were positive to 1:6400 dilution of antisera, showing exceptional reactivity. Working formulation/parameters were 10 µL sample, 100 µL PBS, pH 7.4/PEG6000/detergent and 95 µL R2 (antisera). Sample/R1 was incubated for 5-minutes before addition of R2; reaction was monitored at 340 nm/37°C. LOD and linearity were also assessed. No cross-reactivity with k4t2 or plasminogen up to the concentrations tested was observed. Linearity to 330 nmol/L and LOD of 18 nmol/L were found. Conclusions The use of this antisera/assay eliminates the bias associated with apo(a) size polymorphisms and allows for the accurate measurement of Lp(a) particle number. More work is needed to optimize assay parameters and formulation and comprehensively validate assay performance.

B-291 Validation of a Simple Qualitative Immunoassay for 21 Drugs of Abuse in Meconium on a Single Biochip Array

Abstract Background Drugs use during pregnancy is a major social and medical issue, which may lead to significant pre- and post-natal complications, such as preterm birth and newborn mortality. Exposed newborns may have long-term behavioral, cognitive, and developmental problems. Accurate detection of in utero drug exposure is crucial for timely medical intervention for the newborn and the mother. Meconium is the ‘gold standard' matrix for detection of drugs in newborn which reflects drug exposure during pregnancy. This study aims to develop and optimize a clinical assay for simultaneous screening of 21 drugs in meconium using a custom biochip array. Methods Custom biochip array (Randox Laboratories Ltd.) included 21 discrete test regions (DTRs) for amphetamine, methamphetamine, opioids/opiates (targeting morphine, oxymorphone, oxycodone, hydrocodone, 6-monoacetyl morphine, tramadol, buprenorphine, fentanyl, and methadone), phencyclidine (PCP), cocaine (targeting benzoylecgonine), benzodiazepines (targeting oxazepam, lorazepam, and clonazepam), zolpidem, barbiturates (targeting phenobarbital), ethyl glucuronide, methylphenidate, and marijuana metabolite (targeting Delta-9-Carboxy-Tetrahydrocannabinol [THC-COOH]). Patient samples were obtained from the residual meconium samples that had been previously used for other tests. Blank meconium samples were spiked at in-house cutoff, negative and positive assay control levels. All samples were extracted in specimen diluent using Biotage® Lysera and centrifuged. The supernatant was analyzed on a fully automated Randox Evidence+® Analyzer. A robotic arm pipetted 60 µL sample on to a biochip and then the analyzer performed a competitive chemiluminescent immunoassay. The results were generated as relative light units (RLUs). Results The sample results were determined to be “positive” or “negative” based on average RLU of cutoff controls for each analyte on an assay. The intra-assay (CV ≤ 15%) and inter-assay (CV ≤ 18%) precision was 100% agreement for positive and negative controls and there was no observed carryover for any of the analytes. The sample recovery was excellent with minimal to no matrix suppression for all analytes. Accuracy was performed by comparing spiked analyte results from Randox Evidence+® Analyzer with the expected results and all the spiked samples matched (positive or negative). When authentic patient or spiked results were compared with external laboratory test results, some discordant results were obtained. However, the discrepancies could be explained based on the different cutoffs (e.g., the Randox assay had lower cutoffs) or the known assay cross reactivities within the analytes of same class. These deviations did not affect clinical utility of the assay. Conclusions We validated a novel and simple qualitative chemiluminescent immunoassay in meconium using a custom biochip array. Unlike other routine screening/ELISA methods, which require separate assays for detection of similar drug targets, the custom biochip array offers a convenient, rapid and efficient method that utilizes only a single sample preparation for the screening of all 21 drug targets. Therefore, this technology has potential to be utilized as high-throughput screening assay. Like all qualitative assays, the limitation of this test is that all positive results will need to be assessed quantitatively for a definitive interpretation.

Determination of drugs of abuse and metabolites in plasma microsamples by LC-MS/MS after microQuEChERS extraction.

Aim: Identifying drugs of abuse and their metabolites in plasma is vital in both forensic and clinical toxicology. While the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method offers an efficient approach to sample preparation, its application is complex due to the wide-ranging properties of target analytes and the challenges posed by biological matrix interferences. This study aims to develop a microQuEChERS approach for the quantification of 14 drugs of abuse and metabolites utilizing minimal sample and solvent volumes.Methods: The microQuEChERS method involved using 10μl plasma samples, 25mg of a salt mixture and 150μl of acetonitrile. Extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), with a 7.5min run. The assay was validated according to bioanalytical guidelines.Results: The accuracy was 96.8-112.4%. The within-assay precision was within 2.0-8.9% and the between-assay precision was within 3.2-8.2%. Matrix effects were found to range from -5.7 to 13.5%. The extraction yield was higher than 74.7%.Conclusion: This study described a microQuEChERS sample preparation approach for determining drugs of abuse and metabolites using plasma microsamples and LC-MS/MS. The approach is efficient, environmentally friendly and suitable for scenarios with limited amounts of biological samples.

Development of an Automatic Liquid Dosing System in Microliter Scale

The goal of this study is to design a novel automatic liquid dosing system for liquid sampling at the microliter level. For this purpose, a mechatronics system is designed to position a syringe at the desired position in the workspace and then drive its piston to inject the liquid to be sampled. Then, an application-specific algorithm is developed to be able to prepare samples in 3x3, 4x4, and 5x5 sample container arrays with a desired volume. The performance tests are conducted for preparing samples with up to three different liquids. The repetitive experiments are performed for 50 and 100 µL sampling volumes. The results indicated that it is possible to dose a single liquid with the highest average deviation of 3.9%. Moreover, it is found that it is possible to prepare a sample with a mixture of three liquids by the highest average deviation from the reference value of around 3.4% when the targeting sampling volume is 250 µL for each liquid.

Quantifying non-transferrin-bound iron (NTBI) in human plasma: incorporating BODIPY-pyridylhydrazone (BODIPY-PH) within a thin green film linked to a portable fluorescence-based device.

Free iron in human serum or non-transferrin-bound iron (NTBI) can generate free radicals and lead to oxidative damage. Moreover, it is highly toxic to various tissues and a vital biomarker related to the iron-loading status of thalassemia and Alzheimer's patients. In NTBI in healthy individuals, NTBI levels are typically less than 1µM; current NTBI analysis usually requires advanced instrumentation and many-step sample pretreatment. To address this issue, we employed our invented BODIPY derivative, BODIPY-PH, as a fluorescence probe and trapped it onto the microcentrifuge tube lid using tapioca starch. The fluorescence intensity of BODIPY-PH increased with increasing NTBI concentration (turn-on). The developed portable reaction chamber facilitates rapid analysis (∼5min) using small sample volumes (10 μL sample in a total volume of 600 μL). Under optimum conditions, using the sample-developed portable fluorescence device and fluorescence spectrometer, we achieved impressive limits of detection (LOD) of 0.003 and 0.0015μM, respectively. Furthermore, the developed sensors show relatively high selectivity toward Fe3+ over other metal ions and biomolecules (i.e., Fe2+, Cr3+, Cu2+, and glucose). The sensor performance in serum samples of thalassemia patients exhibited no significant difference compared to the labeled value (obtained from standard methods). Overall, the developed fluorescence sensor is suitable for determining NTBI and offers high sensitivity, high selectivity, and a short incubation time (5min). Moreover, the method requires a limited number of reagents, is simple to use, and uses low-cost equipment to determine NTBI in human serum samples.

Determination of ivermectin in plasma and whole blood using LC-MS/MS.

Ivermectin is a widely used drug for the treatment of helminthiasis and filariasis worldwide, and it has also shown promise for malaria elimination through its potent mosquito-lethal activity. The objective of this study was to develop and validate a high-throughput and sensitive method to quantify ivermectin in plasma and whole blood samples, using automated sample extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Phospholipids were removed in patient whole blood (100 µl) and plasma (100 µl) samples using a 96-well plate Hybrid-solid phase extraction technique. Ivermectin and its isotope-labelled internal standard (ivermectin-D2) were separated on an Agilent Poroshell 120 EC-C18 50mm × 3.0mm I.D. 2.7µm, using a mobile phase of acetonitrile: ammonium formate 2 mM containing 0.5% formic acid (90: 10, v/v). Detection was performed using a triple quadrupole mass spectrometer in the positive ionization mode. The method was validated in the concentration range 0.970 - 384 ng/ml in both plasma and whole blood matrices. Intra- and inter-batch precisions during the validation were below 15%. There was no carryover or matrix effects detected. Ivermectin is a stable compound and results showed no degradation in the different stability tests. The validated method proved to have high sensitivity and precision, good selectivity and to be suitable for clinical application or laboratory quantification of ivermectin in plasma or whole blood samples.

A quantitative method for the analysis of total and unbound concentrations of amoxicillin, ampicillin, ceftazidime, ceftriaxone, ertapenem, fosfomycin and penicillin G in human plasma with liquid chromatography-tandem mass spectrometry assay.

Monitoring antibiotic plasma levels is critical in populations with altered pharmacokinetics, such as critically ill patients in neonatal or adult intensive care units. This study aimed to develop and validate a rapid, reproducible and sensitive liquid chromatography-tandem mass spectrometry assay (LC-MS/MS) for measuring total and unbound concentrations of amoxicillin, ampicillin, ceftazidime, ceftriaxone, ertapenem, fosfomycin and penicillin G in human plasma. The method required 20 and 250 μl sample volumes for measuring total and unbound concentrations, respectively. Sample preparation involved protein precipitation and the addition of an internal standard. Ultrafiltration separated unbound drugs. Method validation covered selectivity, carryover, linearity, accuracy, precision, dilution effects, matrix effects and stability. The LC-MS/MS was performed within a run time of 7.5min. Calibration curves were linear for ceftazidime and ertapenem (ranges 0.1-50 and 0.05-100 mg/l, respectively) and quadratic for other analytes (0.1-50 mg/l, except for ampicillin: 0.1-20 mg/l; R2 > 0.990). Accuracy was within ±15% of the nominal concentration, and precision did not exceed ±15% (relative standard deviation). Samples showed no significant degradation at the tested temperatures and time points. Clinical applicability was demonstrated in a critically ill neonate. This method with minimal sample volume and short analysis time enables the measurement of total and unbound concentrations of selected antibiotics, and is suitable for routine clinical care and studies.

Examining the genomic influence of topically applied probiotics invitro.

Recent work examined the influence of topically applied just-add-water creams containing Lactobacillus plantarum probiotic cultures on to reconstructed human epidermis (RHE). The ability to blend various quiescent probiotic powders in topical systems allows for the examination of these powders on RHEs employing various individual quiescent probiotics using human gene microarrays. Four topical Just-Add-Water powders (STRATABIOSYS™ Technologies) were prepared containing the following: (1) Lactobacillus plantarum Lp90 [200 M CFU/g]; (2) Saccharomyces cerevisiae [200 M CFU/g]; (3) Streptococcus thermophilus [200 M CFU/g]; and (4) Lactococcus Lactis LLa61 [200 M CFU/g]. A powder without probiotics was used as a placebo control. The creams were prepared by taking 3% of each powder and dissolving it into sterile water. A 15 μL sample of each cream was applied to a RHE tissue that presented approximately 90 K CFU/g of each microorganism on the tissue. The RHE was treated for 24 h with the creams whereupon the residual cream was rinsed off, and the tissues were analysed using Agilent human gene microarrays containing 19 217 individual genes from which a smaller subset of 244 genes pertinent to skin were culled. The following probiotic specific gene responses were found: (1) Lactobacillus plantarum upregulated 4.9% of the skin-relevant gene set; (2) Saccharomyces cerevisiae upregulated 7.8% of the skin relevant gene set; Streptococcus thermophilus upregulated 6.1% of the skin relevant gene set; Lactococcus lactis upregulated 7.0% of the skin relevant gene set. A method to examine topical probiotics on RHE has been described that involves converting the powders to preservative-free creams.