Undiluted Samples Research Articles

Flow cytometry for RBC damage and complement activation

Abstract Introduction Red blood cells (RBCs) express anionic phospholipids such as phosphatidylserine (PS) in their inner cell membrane layer. As RBCs age, physiochemical changes in their cell membranes, particularly exposure of PS on the outer membrane layer, lead to phagocytosis and clearance from circulation. PS in RBCs induces activation of the complement system, increasing binding of C3 to RBCs. Thus, quantifying PS-expressing (PS+) RBCs and complement deposition on RBCs (C3+) is a potential measure of RBC injury and may improve transfusion quality when used for predicting post-transfusion RBC recovery. Our goal was to develop a flow cytometry assay that quantifies PS+ and C3+RBCs and to assess how this assay correlates with the direct antiglobulin test for C3 (C3-DAT). Methods Using the Cytoflex S flow cytometer, we measured PS+ and C3+ RBCs using anti-annexin-PE (PS+RBC) and biotin-labeled anti-C3 with streptravidin-PE-Cy5 (C3+RBC). RBCs from healthy donors (NL-RBCs) with negative direct antiglobulin tests for C3 (C3-DAT) (n = 4) were analyzed. To assess the sensitivity of the assay and correlation with C3-DATs, complement control cells (CCC; Immucor) were used as a positive control for both PS+RBCs and C3+RBCs and diluted to different concentrations by mixing CCCs with NL-RBCs. Aliquots of diluted RBCs were analyzed blinded for C3-DAT. Differences between means for PS+RBC and C3+RBC were compared using two-sample t-test, and one-way ANOVA and Tukey’s HSD test were used to determine differences in C3+RBCs among a C3-DAT range of reactivities (0+ to 3+) in diluted CCC samples. P values less than 0.05 were statistically significant. Results In healthy, DAT-negative individuals, the mean positivity for PS+RBCs was 0.28% and for C3+RBCs was 0.34%, indicating that healthy individuals have very low percentages of RBCs with PS and C3 on their cell membrane surface. In CCC diluted samples, the mean C3+RBC percentage was 1.4% in negative C3-DAT samples, 5.9% in weak+ C3-DAT samples, 28.1% in 1+ C3-DAT samples, 67.2% in 2+ C3-DAT samples, and 99.0% in 3+ C3-DAT samples. One-way ANOVA revealed a statistically significant difference in C3+RBCs amongst all C3-DAT reactivities in diluted CCC samples (P<0.001), and Tukey’s HSD test found a mean difference in C3+RBCs percentage of 26.7% between 0+ and 1+ samples (P<0.001), 39.2% between 1+ and 2+ samples (P<0.001), and 31.8% between 2+ and 3+ samples (P<0.01). Undiluted CCC samples had a mean PS+RBC percentage of 7.0% and was statistically different from the PS+RBC percentage in NL-RBCs (P<0.01). Conclusion We describe a feasible assay to measure both PS+RBCs and C3+RBCs using flow cytometry in which CCC can be used as a positive control for PS+RBCs. This assay could serve to evaluate the quality of transfused RBC units and provide insights into the role of phosphatidylserine expression and complement deposition for RBC clearance.

HERBAL-INFUSED EGYPTIAN BEE HONEY: A BOON OR A CURSE? ITS IMPACT ON SENSORIAL, PHYSICOCHEMICAL, & ANTIBACTERIAL PROPERTIES

Herbal honey mixture remedies are well known for their therapeutic benefits in traditional medicine. This research aspired to assess melissopalynological, sensorial, physicochemical, & antibacterial activity for three types of honey (clover, citrus, and cucurbits) and their mixtures with black seed, date palm pollen, & wheat germ at 1, 2.5, & 5%. The best mixtures were chosen according to overall acceptability. Consumer’s preference was given to raw honey. However, some mixtures were as acceptable as raw honey. Honey’s naturalness is characterized by a predominance of fructose over glucose, as revealed by melissopalynological analysis. Herbs infused with discrete kinds of honey typically exhibited higher electrical conductivity, free acidity, ash, H2O2, and HMF levels, but lower pH. The type of honey herb may affect the physicochemical characteristics of honey in different ways. Honey, whether used alone or in combination with the three herbs, demonstrated the same significant antibacterial effect for Staphylococcus aureus and MRSA. Inhibition zones of honey and its mixtures were lower than the control for Pseudomonas aeruginosa. Most undiluted samples created larger bacterial inhibition zones than their 50% diluted counterparts.

Integrated Ultrasound-Enrichment and Machine Learning in Colorimetric Lateral Flow Assay for Accurate and Sensitive Clinical Alzheimer's Biomarker Diagnosis.

The colloidal gold nanoparticle (AuNP)-based colorimetric lateral flow assay (LFA) is one of the most promising analytical tools for point-of-care disease diagnosis. However, the low sensitivity and insufficient accuracy still limit its clinical application. In this work, a machine learning (ML)-optimized colorimetric LFA with ultrasound enrichment is developed to achieve the sensitive and accurate detection of tau proteins for early screening of Alzheimer's disease (AD). The LFA device is integrated with a portable ultrasonic actuator to rapidly enrich microparticles using ultrasound, which is essential for sample pre-enrichment to improve the sensitivity, followed by ML algorithms to classify and predict the enhanced colorimetric signals. The results of the undiluted serum sample testing show that the protocol enables efficient classification and accurate quantification of the AD biomarker tau protein concentration with an average classification accuracy of 98.11% and an average prediction accuracy of 99.99%, achieving a limit of detection (LOD) as sensitive as 10.30pgmL-1. Further point-of-care testing (POCT) of human plasma samples demonstrates the potential use of LFA in clinical trials. Such a reliable lateral flow immunosensor with high precision and superb sensing performance is expected to put LFA in perspective as an AD clinical diagnostic platform.

Evaluation of a Chemiluminescent Enzyme Immunoassay for the Detection of Prostaglandin E-Major Urinary Metabolite (PGE-MUM).

We developed a fully automated quantitative immunoassay for the detection of prostaglandin E-major urinary metabolite (PGE-MUM). In this study, we evaluated the analytical performance of this assay. Sensitivity, within-run reproducibility, correlation with radioimmunoassay (RIA), cross-reactivity, dilution linearity, spike recovery performance, analyte stability, and effects of coexisting substances were evaluated. The assay was also used to measure PGE-MUM in 211 healthy people. The limit of detection and quantification were 1.0 and 1.3 ng/mL, respectively. When the assay was performed six times in a single run, the coefficient of variation ranged from 1.4% to 2.2%. The coefficient of correlation with a preceding RIA method was 0.970 with a correlation slope of 0.88. There was no cross-reactivity with PGE-MUM analogs. Linearity of dilution was confirmed at up to 16-fold dilution with assay results within 100 ± 20% of the theoretical values calculated based on the undiluted sample. Spike recovery was good and ranged from 94% to 101%. Analyte stability was tested by storing samples at 25°C for 6 days, 10°C for 1 month, and by performing up to five freeze-thaw cycles. Assay results were all within 100 ± 10%, the values measured before storage and before the freeze-thaw process. Assay results in healthy people ranged from 3.1 to 162.7 ng/mL (mean: 35.8 ng/mL). After correction for creatinine, the 95% confidence interval was 8.68-42.25 μg/g creatinine. The assay precisely detects PGE-MUM.

Whole Blood Theophylline Measurements Using an Electrochemical DNA Aptamer-based Biosensor

Theophylline, a bronchodilator used in the treatment of respiratory diseases, displays a narrow therapeutic range requiring therapeutic drug monitoring to maximize its effectiveness and safety. Current measurements of theophylline concentrations are, however, achieved using chromatography or immunoassays, which, in spite of their high accuracy and sensitivity, rely on expensive laboratory-based instruments operated by trained personnel or provide only semi-quantitative results, respectively. In response, we developed an electrochemical DNA aptamer-based (E-AB) sensor for theophylline on screen-printed electrodes. Our sensors enable rapid (<30 s) and selective measurements against theophylline structural analogs in clinically relevant range (55–110 μM) in finger-pricked-sized volumes of undiluted blood samples (<100 μL). Given these attributes, we envision that our results contribute to the development of a low-cost and convenient sensing device for molecular monitoring at the point-of-care.

Ecotoxicological assessment of waste foundry sands and the application of different classification systems.

The application of a battery of bioassays is widely recognized as a useful tool for assessing environmental hazard samples. However, the integration of different toxicity data is a key aspect of this assessment and remains a challenge. The evaluation of industrial waste leachates did not initially undergo any of the proposed integration procedures. This research addressed this knowledge gap. Twenty-five samples of waste foundry sands were subjected to a leaching test (UNI EN 12457-2) to evaluate waste recovery and landfill disposal. The leachates were evaluated using a battery of standardized toxicity bioassays composed of Aliivibrio fischeri (EN ISO 11348-3), Daphnia magna (UNI EN ISO 6341), and Pseudokirchneriella subcapitata (UNI EN ISO 8692), both undiluted and diluted. Daphnia magna and P. subcapitata were the most affected organisms, with significant effects caused by 68% and 64% of undiluted samples, respectively. The dilution of samples facilitates the calculation of EC50 values, which ranged from greater than the highest concentration tested to 2.5 g/L for P. subcapitata. The data on single-organism toxicity were integrated using three methods: the Toxicity Classification System, the toxicity test battery integrated index, and the EcoScore system. The three classifications were strongly similar. According to all applied systems, three samples were clearly nontoxic (from iron casting plants) and two were highly toxic (from steel casting plants). Moreover, the similar ranking between undiluted and diluted leachates suggests the possibility of using only undiluted leachates for a more cost-effective and time-efficient screening of waste materials. The findings of this study highlight the usefulness of integrating ecotoxicological waste assessment. Integr Environ Assess Manag 2024;00:1-18. © 2024 The Author(s). Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Label-Free Microfluidic Apheresis of Circulating Tumor Cell Clusters.

Screening liters of blood (i.e., apheresis) represents a generalized approach to promote the reliable access to circulating tumor cell clusters (CTCCs), which are known to be highly metastasis-competent, yet ultrarare. However, no existing CTCC sorting technology has demonstrated high throughput, high yield, low shear stress, and minimal blood dilution simultaneously as required in apheresis. Here, a label-free method is introduced termed Precision Apheresis for Non-invasive Debulking of cell Aggregates (PANDA) to continuously isolate CTCCs from undiluted blood to clean buffer through size sorting, processing 1.4 billion cells per second. The cell focusing is optimized within whole blood leveraging secondary transverse flow and margination. The PANDA chip recovers >90% of spiked ≈24 rare HeLa cell clusters from 100mL undiluted blood samples (equivalent to ≈500 billion blood cells) at 1 Lh-1 throughput, with ≤20s device residence time, ≤15Pa shear stress, and >99.9% return of blood components. The technology lays the groundwork for future routine isolation to increase the recovery of these ultrarare yet clinically significant tumor cell populations from large volumes of blood to advance cancer research, early detection, and treatment.

Interpreting urinary iodine concentration: effects of urine dilution and collection timing.

In population studies, iodine intake estimation relies on median urinary iodine concentration (UIC). However, interpreting UIC measurements can be challenging. In our study, we included 772 adult participants from three groups: nationally representative gender-mixed, women of reproductive age, and pregnant women. We measured UIC and urinary creatinine (U-Cr) to calculate the iodine-to-creatinine ratio (I/Cr). U-Cr cut-off value of 0.226 g/L was used for differentiation between diluted and undiluted urine samples. After excluding samples below this cut-off, new median UIC and I/Cr ratios were calculated. We additionally evaluated the influence of urine sample collection time on UIC. Median UICs were 91.8 µg/L for nationally representative group, 58.3 µg/L for women of reproductive age, and 74.9 µg/L for pregnant women, while I/Cr ratios were 91.7, 102.0, and 159.2 µg/g, respectively. After implementing U-Cr cut-off and excluding all data where U-Cr was below cut-off, new median values were 93.4, 76.3, and 95.4 µg/L for UICs, and 88.6, 88.8, and 128.7 µg/g for I/Cr ratios, respectively. In women of reproductive age, median UIC was significantly lower in urine samples collected after 9:30 and after 12:00 as compared to samples collected before 9.30 (53.4, 57.8, and 97.3 μg/L, respectively). UIC results should be interpreted with caution, considering urine dilution and sample collection timing. U-Cr measurement should be included in population-based iodine intake studies, with corrections applied especially for pregnant women and younger adults, for whom morning is best for single-spot samples.

Laser-Patterning of Micromagnets for Immuno-Magnetophoretic Exosome Capture.

Efficient isolation and patterning of biomolecules is a vital step within sample preparation for biomolecular analysis, with numerous diagnostic and therapeutic applications. For exosomes, nanoscale lipid-bound biomolecules, efficient isolation is challengingdue to their minute size and resultant behavior within biofluids. This study presents a method for the rapid isolation and patterning of magnetically tagged exosomes via rationally designed micromagnets. Micromagnet fabrication utilizes a novel, scalable, and high-throughput laser-based fabrication approach that enables patterning at microscale lateral resolution (<50µm) without lithographic processing and is agnostic to micromagnet geometry. Laser-based processingallows for flexible and tunable device configurations, and herein magnetophoretic capture within both an open-air microwell and an enclosed microfluidic system is demonstrated. Patterned micromagnets enhance localized gradient fields throughout the fluid medium, resulting in rapid and high efficiency magnetophoretic separation, with capture efficiencies nearing 70% after just 1s within open-air microwells, and throughputs upward of 3mLh-1 within enclosed microfluidic systems. Using this microchip architecture, immunomagnetic exosome isolation and patterning directly from undiluted plasma samples is further achieved. Lastly, a FEA-based modeling workflow is introduced to characterize and optimize micromagnet unit cells, simulating magnetophoretic capture zones for a given micromagnet geometry.

Prevalence of Dichelobacter nodosus in western Austrian sheep flocks: Comparison of bacterial cultures, clinical foot rot and lameness with PCR and analysis of sample pooling for PCR diagnosis.

Ovine foot rot is a highly contagious and multifactorial claw disease, caused by Dichelobacter nodosus (D. nodosus) and is the main cause of lameness in sheep. The aim of this cross-sectional study was to determine the prevalence of D. nodosus in western Austria both at animal and farm levels. Real-time PCR was evaluated in comparison with clinical and bacteriological investigations from interdigital foot swabs to detect D. nodosus-infected animals. In addition, the use of pooled four-foot swabs to detect foot rot was determined. In course of the study a total of 3156 sheep from 124 farms were examined for lameness and clinical signs of foot rot. The found flock prevalence of D. nodosus was 30,65 % with bacterial culture showing a sensitivity of 75,0 % and a specificity of 100,0 % (p < 0,001) respectively, compared with PCR. Furthermore, clinical foot rot scores (Ckorr = 0,87; p < 0,001) and lameness scores (Ckorr = 0,71; p < 0,001) highly correlated with the detection of D. nodosus by PCR. The result showed that the clinical examination can be used to identify animals infected with D. nodosus in flocks, but PCR must be used to confirm the diagnosis. D. nodosus could be detected equally well with risk-based pools-of-five samples as with undiluted samples (p < 0,001), suggesting that a pool-of-five samples might be a suitable and cost-effective method for detecting D. nodosus in sheep flocks. This study provides an overview of foot rot in Tyrolean sheep flocks and outlines the possibilities and limitations of the various diagnostic tools for D. nodosus. Further studies to investigate possible influencing factors, including alpine pasturing, management factors and biosecurity predisposing to foot rot are necessary for the design of effective future control programs in alpine regions.

Advanced nanocellulose-based electrochemical sensor for tetracycline monitoring

Antibiotics play a pivotal role in healthcare and agriculture, but their overuse and environmental presence pose critical challenges. Developing sustainable and effective detection methodologies is crucial to mitigating antibiotic resistance and environmental contamination. This study presents a cellulosic polymer-based electrochemical sensor by integrating TEMPO-oxidized cellulose nanofibers-polyethyleneimine hybrids (TOCNFs-PEI) with single-walled carbon nanotube networks (SWCNTs). Our research focuses on (i) conducting physicochemical and electrochemical studies of multifunctional SWCNT/TOCNFs-PEI architectures, (ii) elucidating the relationships between the material's properties and their electrochemical performance, and (iii) assessing its performance in detecting tetracycline concentrations in both controlled and more complex matrices (treated wastewater effluents). The limits of detection were evaluated to be 0.180 µmol L−1 (at the potential of 0.85 V) and 0.112 µmol L−1 (at the potential of 0.65 V) in phosphate-buffered saline solution, and 2.46 µmol L−1 (at the potential of 0.82 V) and 1.5 µmol L−1 (at the potential of 0.65 V) in the undiluted membrane bioreactor effluent sample, respectively. Further, the designed cellulosic polymer-based sensing architecture is compatible with large-scale production, paving the way for a new era of green, versatile sensing devices. These developments will significantly contribute to global efforts to alleviate antibiotic resistance and environmental contamination.

IL-9 is a Biomarker of BIA-ALCL Detected Rapidly By Lateral Flow Assay.

A delayed seroma around breast implants is the most common clinical presentation of BIA-ALCL. However, most seromas are due to benign causes. Therefore, it is essential to distinguish benign seromas from seromas due to BIA-ALCL. In a prior study mean concentrations of IL-9, IL-10 and IL-13 were found to be significantly higher in BIA-ALCL than in benign seromas. The aim of this research was to test the ability to detect high concentrations of IL-9 rapidly with a lateral flow assay (LFA). Because we previously reported that a LFA for CD30 detected BIA-ALCL in seromas we compared CD30 and IL-9 LFAs in distinguishing BIA-ALCL from benign seromas. Thirty microliter samples of 26 seromas (15 benign, 11 malignant) were tested on in-house prepared strips for IL-9 and CD30. Nanoparticle-conjugated antibodies specific to IL-9 and CD30 were used for detection. IL-9 was analyzed in undiluted samples and CD30 samples were optimized at 1:3 dilution. The dynamic range of detection was determined by spiking recombinant IL-9 into a benign seroma. Image analysis measured intensity of both test line (TL) and control line (CL) and a TL/CL ratio was calculated. IL-9 protein and IL-9 transcription factor PU.1 were stained in BIA-ALCL lines and clinical samples. The IL-9 LFA was reliable in distinguishing BIA-ALCL from benign seromas when the concentration of IL-9 was greater than 10 ng/ml. The CD30 LFA was positive in all 11 malignant cases. In one case with only faint CD30 and IL-10 test lines, the IL-9 LFA was clearly positive. Immunohistochemistry showed IL-9 and its essential transcription factor PU.1 were present in tumor cells in BIA-ALCL lines and clinical samples. IL-9 is a tumor cell biomarker of BIA-ALCL that can be detected by lateral flow assay and immunohistochemistry. Concentrations of IL-9 greater than 10 ng/ml reliably distinguished BIA-ALCL from benign seromas. Moreover, IL-9 LFA could detect BIA-ALCL when CD30 LFA was not definitive and IL-10 was of low concentration with a faint IL-10 TL, suggesting a multiplex LFA including IL-9, CD30 and IL-10 might be more effective in detecting BIA-ALCL in selected cases.

Quantitatively measuring the cytotoxicity of viscous hydrogels with direct cell sampling in a micro scale format “MicroDrop” and its comparison to CCK8

Tissue engineering holds promise for developing therapeutic applications using viscous materials e.g. hydrogels. However, assessing the cytotoxicity of such materials with conventional assays can be challenging due to non-specific interactions. To address this, we optimized a live/dead staining method for quantitative evaluation and compared it with the conventional CCK8 assay. Our MicroDrop method involved seeding droplets containing 5000 cells in 10 µl medium on 12-well plates. After allowing them to adhere for 4 h, various viscous samples were applied to the cells and measurements were conducted using a fluorescence microscope immediately and at daily intervals up to 72 h. A sodium dodecyl sulfate (SDS) dilution series compared the MicroDrop with the CCK8 assay. The findings revealed a cell-type specific pattern for 10 mg/ml hyaluronic acid (HA), wherein MC3T3-E1 cells maintained 95% viability until 72 h, while L929 cells experienced a gradual decline to 17%. 2 mg/ml HA exhibited consistent viability above 90% across all time points and cell lines. Similarly, fibrin demonstrated 90% viability across dilutions and time points, except for undiluted samples showing a decrease from 85% to 20%. Gelatin-methacrylol sustained viability above 70% across all time points at both 5% and 10% concentrations. The comparison of the SDS dilution series between viability (MicroDrop) and metabolic activity (CCK8) assay showed a correlation coefficient of 0.95. The study validates the feasibility of the established assay, providing researchers with an efficient tool for assessing cytotoxicity in viscous materials. Additionally, it holds the potential to yield more precise data on well-known hydrogels.Graphical

Comparative Performance Evaluation of Double-Stranded RNA High-Throughput Sequencing for the Detection of Viral Infection in Temperate Fruit Crops.

There is limited information on the compared performances of biological, serological. and molecular assays with high-throughput sequencing (HTS) for viral indexing in temperate fruit crops. Here, using a range of samples of predetermined virological status, we compared two performance criteria (inclusivity and analytical sensitivity) of enzyme-linked immunosorbent assay (ELISA), molecular hybridization, reverse transcription (RT)-PCR, and double-stranded RNA (dsRNA) HTS for the detection of a total of 14 viruses (10 genera) and four viroids (three genera). When undiluted samples from individual plants were used, ELISA had the lowest performance, with an overall detection rate of 68.7%, followed by RT-PCR (82.5%) and HTS (90.7%; 100% if considering only viruses). The lower performance of RT-PCR reflected the inability to amplify some isolates as a consequence of point mutations affecting primer-binding sites. In addition, HTS identified viruses that had not been identified by other assays in nearly two-thirds of the samples. Analysis of serial dilutions of fruit tree samples allowed comparison of analytical sensitivities for various viruses. ELISA showed the lowest analytical sensitivity, but RT-PCR showed higher analytical sensitivity than HTS for most of the samples. Overall, these results confirm the superiority of HTS over biological indexing in terms of speed and inclusivity and show that while the absolute analytical sensitivity of RT-PCR tends to be higher than that of HTS, PCR inclusivity is affected by viral genetic diversity. Taken together, these results make a strong case for the implementation of HTS-based approaches in fruit tree viral testing protocols supporting quarantine and certification programs.

Flow cytometer for a dilution-free measurement approach with sample recollection.

Blood testing using flow cytometry is a common and rapid method for the initial screening and diagnosis of patients. Measurements are often combined with other scientific techniques, and analyzed samples are commonly diluted and discarded afterward. When the sample is recollected instead, sample dilution is a challenge when the sample is intended or needed for additional measurements. Therefore, it is advantageous to recollect the undiluted sample. In order to enable measurements of the same undiluted sample aliquot, we designed and constructed a purpose-built flow cytometer. Our instrument employs syringes, acoustic focusing, and an open fluidics system to recollect and reuse the unadulterated sample. The cytometer is compact, which reduces sample consumption. It acquires forward, sideward, and fluorescence signals, offering opportunities for diverse measurement approaches. In particular, our cytometer has been designed to be ready for additional downstream analysis of cells, e.g., applying mass spectrometry, magnetic resonance spectroscopy, or other analytical tools. This study presents results on instrument performance, a comparison with a cytometer that uses standard hydrodynamic focusing, and a proof of concept for multiple measurements.

Vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor in diabetic and non-diabetic retinopathy: associated factors and anatomical correlation

BackgroundThis study aims to investigate the factors affecting the vitreous levels of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VGEF) among patients with pars plana vitrectomy (PPV). Also, this study correlates the levels of PEDF with RRD characteristics.MethodsAll patients who were scheduled for PPV for any indication were included in the study. They were divided into a case group which included patients with advanced PDR and a control group which included the remaining diagnoses. During the PPV, an undiluted vitreous sample was taken and the enzyme-linked immunosorbent assay method was utilized to measure the levels of VEGF and PEDF.ResultsEighty eyes were involved. Patients diagnosed with advanced PDR and endophthalmitis exhibited higher levels of VEGF. PEDF was affected inversely by the age of the patients and PEDF levels were higher in RRD and endophthalmitis cases. In patients with RRD, the level of PEDF was higher if the tear was found inferiorly, if the macula was detached, and with a longer duration of RRD.ConclusionsThis study highlights the clinical importance of those biomarkers. Anti-VEGF-based treatment is the mainstay against PDR. PEDF may show a promising predictive values regarding patients with RRD.

Rapid label-free impedimetric detection of progesterone enhanced by immunomagnetic bead-based competitive immunoreaction

Detecting progesterone (P4) concentration in cow serum is essential for monitoring the pregnancy progress after fertilization and is significant for the dairy farming industry and veterinary medicine. This study reports enzyme-free immunomagnetic beads (IMBs)-based competitive immunoassay for detecting P4 by P4-bovine serum albumin (BSA)-modified biosensors. The anti-P4 antibody-conjugated IMBs serve as collectors to capture P4 in undiluted serum samples to prevent the biosensor surface from biosample contamination and as insulated labels to report the electron-transfer resistance signal of electrochemical impedance spectroscopy (EIS) measurement. The IMBs and P4-containing samples were mixed for 15−30 min, capable of obtaining stable P4@IMB complexes. The 0.2-kGauss pulsed magnetic field (PMF) of the 20-s pulse width and 20-s relaxation time applied for 5 min can shorten the immunoreaction time between the P4@IMBs and the P4-BSA-modified biosensor and reduce the IMB's nonspecific adsorption on the biosensor surface. This competitive immunoassay's cut-off value and detection limit were 7.71 ng/mL and 7.33 ng/mL, respectively, which is lower than the serum's P4 plateau concentration (over 8 ng/mL) of dairy cows on days 6–16 of estrus cycles and that in pregnancy. The IMB-based immunoassay combining the PMF attraction and the label-free EIS measurement exhibits promising potential for rapidly detecting P4 in undiluted serum.

Biomimetic anti-fouling interface engineering based on thorn-vine-like structure for electrochemical sensing

Electrochemical sensing interfaces face an enormous obstacle posed by the non-specific adsorption in complex matrixes. This will decrease the current and sensitivity of electrochemical sensors. Since most biomarkers, especially those associated with diseases such as cancer, exist in trace amounts, sensitive detection is essential for them. However, it is a considerable challenge to develop anti-fouling interface strategies that can achieve highly sensitive electrochemical sensing in complex matrixes. Herein, a novel biomimetic anti-fouling interface engineering strategy was constructed based on thorn-vine-like structure for electrochemical biosensing. Bovine serum albumin (BSA) and sulfobetaine methacrylate (SBMA) underwent a thiol-ene click reaction, followed by electrostatic interaction with multi-walled carbon nanotubes (MWCNT), resulting in the thorn-vine-like structure BSA-PSBMA-MWCNT (BPSMC), with a highly three-dimensional network structure. The BPSMC not only significantly reduced non-specific adsorption, but also effectively enhanced the ability of the sensing interface to transfer electrons to the electrode surface. The impedance change ratio of the BPSMC-modified glassy carbon electrodes (GCE) was only 5.30% compared to the bare GCE when treated in undiluted serum samples for 2 h. As a proof of concept, the tumor biomarker ferritin (FER) was applied as a model analyte. By functionalizing the interface with specific antibodies, it enables quantification of FER in undiluted serum samples with ultra-high sensitivity. This biomimetic anti-fouling interface engineering strategy possesses excellent anti-fouling property, sensitivity, and universality and offers great potential for anti-fouling interfaces in complex matrices for medical and non-medical applications.

Ion transfer mediated by TEMPO in ionophore-doped thin films for multi-ion sensing by cyclic voltammetry

We report here on the development of thin-layer ion-selective membranes containing lipophilic TEMPO as a phase-transfer redox mediator for the simultaneous detection of non-redoxactive ions. This redox probe was recently introduced by our group and provides ideal ion-transfer waves when the membrane is interrogated by cyclic voltammetry. To perform multianalyte detection in the same sensing film, plasticized PVC-based membranes were doped with lithium and potassium ionophores in addition to a lipophilic cation-exchanger. The ionophores allow for ion discrimination owing to the different ionophore-cation complexation constants and the oxidation of TEMPO to the oxoammonium form results in the selective transfer of lithium and potassium at different potentials. The resulting voltammograms have half-peak widths of 100 and 102 mV, and the peak separation between anodic and cathodic scans is 8 and 9 mV for lithium and potassium, respectively, close to theoretical expectations. High peak resolution was observed, and the ion-transfer waves are still distinguishable when the ion activities differ by three orders of magnitude. These parameters are remarkably better than those obtained with other redox probes, which is important for multianalyte detection in the same voltammetric scan. Optimized membranes showed independent Nernstian shifts (slopes of 59.23 mV and 54.8 mV for K+ and Li+, respectively) of the peak position for increasing ion concentrations. An idealized model for two ionophore-based membranes combining redox and phase-boundary potentials was applied to the proposed system with excellent correlation. Potassium and lithium ions were simultaneously detected in undiluted human serum samples with good accuracy and precision.

The lupus anticoagulant titer is associated with elevated antiphosphatidylserine/prothrombin immunoglobulin-M isotype antibody levels.

Clot based assays used for lupus anticoagulant (LAC) detection are typically interpreted in a qualitative fashion and may not reflect LAC potency. In this cross-sectional study, we describe a method for quantifying the LAC titer using serial (dependent) two-fold dilutions in normal pooled plasma. Serial dilutions of 51 residual plasma samples from 50 patients were tested using the Russell's viper venom screening time (DRVVT) and activated partial thromboplastin screening time (APTT) methodologies. The measured clotting times and the corresponding dilution factors were then used to derive a four-parameter logistic model. The LAC titer for each patient was interpolated as the sample dilution that corresponds to the upper reference interval limit of the corresponding assay. Calculated APTT and DRVVT LAC titers displayed a strong linear correlation (R2 = 0.84) between each other, but not with the degree of prolongation of the APTT/DRVVT screening time in the neat undiluted samples. Using data driven partitioning, patients could be grouped into low (<10) or high (≥10) DRVVT LAC titer. There were no significant differences in anticardiolipin (aCL) or anti-beta 2 glycoprotein 1 (aB2GPI) antibody levels or prevalence of thromboembolic events between low and high LAC titer groups. In contrast, antiphosphatidylserine/prothrombin (aPS/PT) IgM antibody levels, but not IgG, were significantly higher in the high LAC titer group. The degree of prolongation of the APTT/DRVVT screening time is not correlated with the LAC titer. Only aPS/PT IgM antibodies levels were strongly correlated with the LAC titers. Additional studies are warranted to determine clinical implications of high LAC titers.