Low Sample Volume Research Articles

Spatial barcoding-enabled highly multiplexed immunoassay with digital microfluidics

Digital microfluidics (DMF), facilitating independent manipulation of microliter samples, provides an ideal platform for immunoassay detection; however, suffering limited multiplexity. To address the need, herein we described a digital microfluidics (DMF) platform that realizes spatial barcoding on the Teflon-coated indium tin oxide (ITO) glass side to fulfill highly multiplexed immunoassay (10+) with low-volume samples (∼4 μL) in parallel, representing the highest multiplexing recorded to date for DMF-actuated immunoassay. Planar-based spatial immobilization of multiple capture antibodies was realized on a Teflon-coated ITO glass side, which was then used as the top plate of the DMF device. Droplets containing analytes, secondary antibodies, and fluorescent signaling reporters with low volume, which were electrically manipulated by our DMF control system, were shuttled sequentially along the working electrodes to complete the immuno-reaction. Evaluation of platform performance with recombinant proteins showed excellent sensitivity and reproducibility. To test the feasibility of our platform in analyzing multiplex biomarkers of the immune response, we used lipopolysaccharide-stimulated macrophages as a model system for protein secretion dynamics studies. As a result, temporal profiling of pro-inflammatory cytokine secretion dynamics was obtained. The spatial barcoding strategy presented here is easy-to-operate to enable a more comprehensive evaluation of protein abundance from biological samples, paving the way for new opportunities to realize multiplexity-associated applications with the DMF platform.

Silver Nanostructured Substrates in LDI-MS of Low Molecular Weight Compounds.

Mass spectrometric techniques can provide data on the composition of a studied sample, utilizing both targeted and untargeted approaches to solve various research problems. Analysis of compounds in the low mass range has practical implications in many areas of research and industry. Laser desorption ionization techniques are utilized for the analysis of molecules in a low mass region using low sample volume, providing high sensitivity with low chemical background. The fabrication of substrates based on nanostructures to assist ionization with well-controlled morphology may improve LDI-MS efficiency for silver nanoparticles with plasmonic properties. In this work, we report an approach for the preparation of silver nanostructured substrates applied as laser desorption ionization (LDI) plates, using the chemical vapor deposition (CVD) technique. Depending on the mass of used CVD precursor, the approach allowed the synthesis of LDI plates with tunable sensitivity for various low molecular weight compounds in both ion-positive and ion-negative modes. Reduced chemical background and sensitivity to small biomolecules of various classes (fatty acids, amino acids and water-soluble metabolites) at nanomolar and picomolar detection levels for lipids such as triacylglycerols, phosphatidylethanolamines and lyso-phosphatidylcholines represent an emerging perspective for applications of LDI-MS plates for the collection of molecular profiles and targeted analysis of low molecular weight compounds for various purposes.

Ultrasensitive multiplexed chemiluminescent enzyme-linked immunosorbent assays in 384-well plates

We have developed an ultrasensitive multiplexed immunoassay using 384-well microtiter plates capable of detecting proteins at subfemtomolar concentrations that requires as little as 2.5 μL of sample. Arrays of up to 4 capture antibodies were patterned on the bottom of the wells of a 384-well plate either by directly printing the capture antibodies or by printing anti-peptide tag anchor antibodies and incubating these arrays with capture antibodies conjugated to the corresponding peptide tags (“customized” assays). Samples were incubated with the antibody arrays and shaken orbitally at 2000 rpm to achieve the greatest sensitivity. Chemiluminescence (CL) from immunocomplexes labeled with horseradish peroxidase was imaged across the entire plate to quantify the amount of protein bound to each antibody spot of the arrays. The 384-well assay had a throughput 5-fold greater than 96-well plates that was achieved from simultaneous imaging of CL in all 384-wells and the use of automated pipettors to allow parallel processing of 384 assays. We developed 4 assays based on the 384-well CL ELISA: a direct print assay for IL-10 (limit of detection (LOD) = 0.075 fM); a customized assay for IL-6 (0.22 fM); a customized pharmacokinetic (PK) assay for measuring adalimumab (7.3 pg/mL); and a customized 4-plex assay for IL-5 (0.1 fM), IL-6 (0.52 fM), IL-10 (0.2 fM), and TNF-α (3.2 fM). The sensitivity and precision of the cytokine assays were comparable to current ultrasensitive protein detection methods in 96-well formats. The PK assay for adalimumab was 650 times more sensitive than a commercially available 96-well plate ELISA. We used the 384-well CL ELISAs to measure endogenous levels of the cytokines in the serum and plasma of healthy humans: the mean concentrations and precision were comparable to those from 96-well immunoassays. This 384-well format with subfemtomolar sensitivity will enable ultrasensitive multiplexed immunoassays to be performed with higher throughput and lower sample volumes than currently possible, a particularly important capability for clinical studies in drug development.

P-388 Isolation and characterization of human uterine fluid lavage-derived extracellular vesicles by three different methods for optimal embryo transfer timing discovery

Abstract Study question What is the suitable method for isolating extracellular vesicles (EVs) from a small volume of Uterine Fluid Lavage (UFL)? Summary answer It seems that the qEV column can help in faster, precise, and gentle isolation of EVs from small amounts of UFL. What is known already Several studies have discovered that existing EVs in the UFL, which are informative carriers, reflect cytological and molecular conditions of the uterine environment. The relative amounts of the EV components are different in various physiological situations. So, they are the ideal source for precise molecular analysis of the uterine condition to identify the Window of Implantation (WOI) for appropriate embryo transfer (ET) timing. There is limited information describing the most efficient method for EV isolation from a low-volume sample of biological fluids. In particular, EV separation from some biological fluids such as UFL is more complicated because of the viscosity. Study design, size, duration The study was conducted in two steps; first, the pre-implantation UFL sampling effect was studied on the implantation and clinical pregnancy rates. UFLs were obtained from 20 IVF Frozen Embryo Transfer (FET) patients as the case group. The control group consisted of 20 FET patients without lavage. In the second step, UFLs (n = 30) were collected and pooled. The EVs were extracted by ultracentrifugation (UC), Sucrose cushion (Suc), and Size-exclusion chromatography (by the qEV column) methods. Participants/materials, setting, methods First, UFL was collected two days before frozen blastocyst ET from the case group using an intrauterine insemination catheter attached to a syringe. Then, patients were compared with control at the seventh week. Second, EVs were extracted from pooled UFL samples and characterized by Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), and Western Blot (WB). Albumin contamination was detected by SDS-PAGE. Total RNA was extracted, and Real-time PCR was performed for six housekeeping genes. Main results and the role of chance A comparison between case and control groups indicated no significant difference for implantation and clinical pregnancy rates. Moreover, we have confirmed the presence of EVs in UFL that were successfully isolated, by the UC, Suc, the qEV methods. The SEM images showed that the collected vesicles were morphologically spherical. The DLS analysis revealed that the intensity peak of isolated particles with the UC and Suc methods were 281 nm and 216 nm, respectively. Extracted EVs using the qEV technique were smaller and homogenized in size, with an intensity peak of 67.7 nm. SDS-PAGE of EVs showed a weaker albumin band in the qEV column (with a 100 kDa filter) method than the UC, Suc, and qEV column (with a 30 kDa filter) methods. Although, their quantification showed no significant differences. WB analysis indicated common EV markers (D81, CD9, CD63, and TSG101) showed better expression using the qEV extraction technique. But CD9 and TSG101 had lower expression levels in EVs isolated using Suc and UC, respectively. Calnexin was not detected in the isolated EVs. The GAPDH, 18SrRNA, ACTIN, GUSB, HMBS, and YWHAZ genes were detected by real-time PCR in all three EV extraction methods. The 18SrRNA gene showed better CT values. Limitations, reasons for caution Uterine fluid samples collection from qualified study groups and the patient’s follow-up for ensuring successful implantation and clinical pregnancy are time-consuming. The low volume of uterine fluid lavage sample and consequently the low number of extracellular vesicles inside it was also the limitation of this study. Wider implications of the findings Endometrial receptivity assay is difficult to study non-invasively. Herein, endometrial secretome EVs as a minimally invasive source for uterine environment molecular assessment is presented. High-throughput analysis of EV components can offer biomarkers for individual determination of ET timing, providing an additional method to use with transvaginal-ultrasonography for assessing endometrial receptivity. Trial registration number not applicable

Plasma protein biomarker profiling reveals major differences between acute leukaemia, lymphoma patients and controls

Aiming to accommodate the unmet need for easily accessible biomarkers with a focus on biological differences between haematological diseases, the diagnostic value of plasma proteins in acute leukaemias and lymphomas was investigated. A multiplex proximity extension assay (PEA) was used to analyze 183 proteins in diagnostic plasma samples from 251 acute leukaemia and lymphoma patients and compared with samples from 60 healthy controls. Multivariate modelling using partial least square discriminant analysis revealed highly significant differences between distinct disease subgroups and controls. The model allowed explicit distinction between leukaemia and lymphoma, with few patients misclassified. Acute leukaemia samples had higher levels of proteins associated with haemostasis, inflammation, cell differentiation and cell-matrix integration, whereas lymphoma samples demonstrated higher levels of proteins known to be associated with tumour microenvironment and lymphoma dissemination. PEA technology can be used to screen for large number of plasma protein biomarkers in low µL sample volumes, enabling the distinction between controls, acute leukaemias and lymphomas. Plasma protein profiling could help gain insights into the pathophysiology of acute leukaemia and lymphoma and the technique may be a valuable tool in the diagnosis of these diseases.

Development of a microfluidic device for low volume sample lysis

Development of a microfluidic device for low volume sample lysis

Applications of Microfluidics and Organ-on-a-Chip in Cancer Research

Taking the life of nearly 10 million people annually, cancer has become one of the major causes of mortality worldwide and a hot topic for researchers to find innovative approaches to demystify the disease and drug development. Having its root lying in microelectronics, microfluidics seems to hold great potential to explore our limited knowledge in the field of oncology. It offers numerous advantages such as a low sample volume, minimal cost, parallelization, and portability and has been advanced in the field of molecular biology and chemical synthesis. The platform has been proved to be valuable in cancer research, especially for diagnostics and prognosis purposes and has been successfully employed in recent years. Organ-on-a-chip, a biomimetic microfluidic platform, simulating the complexity of a human organ, has emerged as a breakthrough in cancer research as it provides a dynamic platform to simulate tumor growth and progression in a chip. This paper aims at giving an overview of microfluidics and organ-on-a-chip technology incorporating their historical development, physics of fluid flow and application in oncology. The current applications of microfluidics and organ-on-a-chip in the field of cancer research have been copiously discussed integrating the major application areas such as the isolation of CTCs, studying the cancer cell phenotype as well as metastasis, replicating TME in organ-on-a-chip and drug development. This technology’s significance and limitations are also addressed, giving readers a comprehensive picture of the ability of the microfluidic platform to advance the field of oncology.

Citrate-Capped Gold Nanoparticle as a Ligand for Chromium(III) Ion: Optimization and Its Application in Contaminated Tap Water

Citrate-capped gold nanoparticle (GNP) was used as a ligand for chromium chelating, and chromium ions reaction led to GNP aggregation. The color change of GNP by aggregation in the presence of chromium is a simple and rapid colorimetric test for these ions in an aqueous solution. GNP capped citrate was prepared by the citrate method and characterized by TEM, and its particle size was 20 nm. Also, the surface plasmon resonance (SPR) peak was 520 nm. In the presence of chromium ions, the color of GNP at 520 nm was shifted to 650 nm because of aggregation to give a signal as a ratio of A650/520 more than one and proportional with chromium concentration directly. The optimum conditions were studied to obtain the high signal represented by the volume of GNP, reaction kinetic of A650 with time, selectivity, and interferences of Zn(II), Fe(III), Fe(II), Sn(II), Ni(II), Ca(II), Al(III), Sr(II), Cu(II), Mn(II), Co(II), Mg(II), Ag(I), and Pb(II) ions. The calibration curve is linear in the range of 100–500 ppb, and the regression was 0.9951 and applied on tap water chromium ions in the same range in the regression of 0.95. This method was simple, rapid reaction, consumed low volumes of sample, and had low detection limits. It can be recommended as a new method for chromium (III) detection in aqueous solutions.

Abstract 2226: Using functional biomarkers to accurately predict advanced neoplasia in pancreatic cystic lesions

Abstract Background: The accurate identification of high-grade dysplasia (HGD) or invasive cancer (advanced neoplasia, AN) in pancreatic cystic lesions (PCLs) is needed to identify PCLs that warrant surgical intervention. Cytologic evaluation of cyst fluid is widely-used but has poor sensitivity. We previously found that activity of the lysosomal serine protease tripeptidyl peptidase 1 (TPP1) is associated with mucinous PCLs that harbor AN (AUC 0.72). We aimed to identify additional functional biomarkers that would improve performance of our classifier for dysplastic grade in mucinous PCLs. Methods: We used a combination of global protease activity profiling and shotgun proteomics to identify differentiating markers between PCLs with low-grade dysplasia (LGD, n=3) and HGD (n=3). Candidate biomarkers were validated in an independent cohort of 28 clinically-annotated mucinous PCLs (HGD n=12, LGD n=16), by measuring both protein concentration (using ELISA) and enzymatic activity (using internally-quenched fluorescent substrates). We used a nested cross-validation approach to iteratively split our cohort into training and validation sets to predict HGD. Model accuracy was evaluated using area under the curve (AUC), sensitivity, specificity, and accuracy. Results: We identified 8 proteins that were highly abundant in mucinous PCLs with HGD. Among these were the inflammatory enzymes myeloperoxidase (MPO) and neutrophil elastase (ELANE), suggesting that differential activity levels could be exploited that would minimize fluid requirements and cost. Among 28 mucinous PCLs, a composite score that included both activity and mass of MPO performed the best for HGD in mucinous cysts (AUC 0.955, sensitivity 98%, specificity 93%). ELANE activity (AUC 0.76, sensitivity 69%, specificity 59%) and TPP1 activity (AUC 0.765, sensitivity 71%, specificity 74%) had modest performance. Conclusions: Our functional biomarkers accurately classified HGD among mucinous cysts. Functional biomarkers that require low sample volumes (<10μl) of cyst fluid have the potential to substantially improve clinical decision-making for patients with PCLs. Citation Format: Francesco Caiazza, Andre Lourenco, Patricia Conroy, Thomas Hoffmann, Sam L. Ivry, Tyler York, Gina Zhu, Audrey Mustoe, Anthony J. O'Donoghue, Charles S. Craik, Kimberly Kirkwood. Using functional biomarkers to accurately predict advanced neoplasia in pancreatic cystic lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2226.

Bridging the Gap between Digital Assays and Point-of-Care Testing: Automated, Low Cost, and Ultrasensitive Detection of Thyroid Stimulating Hormone.

Medical diagnostics is moving toward disease-related target detection at very low concentrations because of the (1) quest for early-stage diagnosis, at a point where only limited target amounts are present, (2) trend toward minimally invasive sample extraction, yielding samples containing low concentrations of target, and (3) need for straightforward sample collection, usually resulting in limited volume collected. Hence, diagnostic tools allowing ultrasensitive target detection at the point-of-care (POC) are crucial for simplified and timely diagnosis of many illnesses. Therefore, we developed an innovative, fully integrated, semi-automated, and economically viable platform based on (1) digital microfluidics (DMF), enabling automated manipulation and analysis of very low sample volumes and (2) low-cost disposable DMF chips with microwell arrays, fabricated via roll-to-roll processes and allowing digital target counting. Thyroid stimulating hormone detection was chosen as a relevant application to show the potential of the system. The assay buffer was selected using design of experiments, and the assay was optimized in terms of reagent concentration and incubation time toward maximum sensitivity. The hydrophobic-in-hydrophobic microwells showed an unparalleled seeding efficiency of 97.6% ± 0.6%. A calculated LOD of 0.0013 μIU/mL was obtained, showing the great potential of the platform, especially taking into account the very low sample volume analyzed (1.1 μL). Although validation (in biological matrix) and industrialization (full automation) steps still need to be taken, it is clear that the combination of DMF, low-cost DMF chips, and digital analyte counting in microwell arrays enables the implementation of ultrasensitive and reliable target detection at the POC.

Particle-Based Microrheology As a Tool for Characterizing Protein-Based Materials.

Microrheology based on video microscopy of embedded tracer particles has the potential to be used for high-throughput protein-based materials characterization. This potential is due to a number of characteristics of the techniques, including the suitability for measurement of low sample volumes, noninvasive and noncontact measurements, and the ability to set up a large number of samples for facile, sequential measurement. In addition to characterization of the bulk rheological properties of proteins in solution, for example, viscosity, microrheology can provide insight into the dynamics and self-assembly of protein-based materials as well as heterogeneities in the microenvironment being probed. Specifically, passive microrheology in the form of multiple particle tracking and differential dynamic microscopy holds promise for applications in high-throughput characterization because of the lack of user interaction required while making measurements. Herein, recent developments in the use of multiple particle tracking and differential dynamic microscopy are reviewed for protein characterization and their potential to be applied in a high-throughput, automatable setting.

PATH-11. Detection of genetic and epigenetic alterations in Liquid Biopsies from pediatric brain tumor patients

Abstract BACKGROUND: Liquid biopsies (LBs) hold great promise as a non-invasive method for tumor detection and disease monitoring. Their application in pediatric neuro-oncology patients has been challenging due to the blood-brain barrier limiting the amount of circulating tumor DNA (ctDNA) and low volumes of cerebrospinal fluid (CSF) and serum samples. Here, we assessed the feasibility of LBs in a pediatric brain tumor cohort (n=12 patients) by adapting pre-analytical protocols to low cell-free DNA (cfDNA) input and bioinformatic pipelines for genome-wide analyses. METHODS: Genomic DNA isolated from tumor tissues, including medulloblastoma (n=6), ependymoma (n=4), low-grade glioma (n=1), and choroid plexus papilloma (n=1), and cfDNA isolated from matched CSF and serum samples were subjected to low-coverage whole genome sequencing (lcWGS) and the EPIC DNA methylation array. For methylation analyses, samples were prepared using both traditional bisulfite- and recently developed enzymatic-based methylation conversion. Molecular tumor (sub)type predictions were performed using the Heidelberg Brain Tumor Classifier. RESULTS: lcWGS revealed tumor-specific copy number variations (CNVs) in both CSF and serum samples. The detection rate of tumor-specific CNVs (median 92% vs. 23%) and the ctDNA fraction (median 31% vs. 1.5%) were higher in CSF vs. serum samples. Bisulfite-converted as well as enzymatically converted tumor DNA and cfDNA samples could be readily analyzed using the Heidelberg Classifier. In comparison to bisulfite-based conversion, enzymatic-based conversion improved the quality of methylation analyses for minimal input cfDNA samples. CONCLUSIONS: Our results highlight the feasibility of liquid biopsies from both CSF and serum in brain tumor patients. In contrast to CNV-based tumor detection from serum samples, ctDNA analysis from CSF allowed a more comprehensive genetic and epigenetic profiling which is expected to assist in less-invasive tumor classification, identification of drug targets, or the investigation of tumor evolution. Altogether, this study provides the rationale for further implementation of LBs in pediatric neuro-oncology.

Combining Machine Learning and Backgrounded Membrane Imaging: A Case Study in Comparing and Classifying Different Types of Biopharmaceutically Relevant Particles

This study investigates how backgrounded membrane imaging (BMI) can be used in combination with convolutional neural networks (CNNs) in order to quantitatively and qualitatively study subvisible particles in both protein biopharmaceuticals and samples containing synthetic model particles. BMI requires low sample volumes and avoids many technical complications associated with imaging particles in solution, e.g., air bubble interference, low refractive index contrast between solution and particles of interest, etc. Hence, BMI is an attractive technique for characterizing particles at various stages of drug product development. However, to date, the morphological information encoded in brightfield BMI images has scarcely been utilized. Here we show that CNN based methods can be useful in extracting morphological information from (label-free) brightfield BMI particle images. Images of particles from biopharmaceutical products and from laboratory prepared samples were analyzed with two types of CNN based approaches: traditional supervised classifiers and a recently proposed fingerprinting analysis method. We demonstrate that the CNN based methods are able to efficiently leverage BMI data to distinguish between particles comprised of different proteins, various fatty acids (representing polysorbate degradation related particles), and protein surrogates (NIST ETFE reference material) only based on BMI images. The utility of using the fingerprinting method for comparing morphological differences and similarities of particles formed in distinct drug products and/or laboratory prepared samples is further demonstrated and discussed through three case studies.

Relative viscosity evaluation of low-volume samples using capillary flow under differential pressure

In this study, we newly considered the analysis of capillary flow under differential pressure, which extends the previous study and allows the independent measurement of relative viscosity and the product of relative surface tension and contact angle for low-volume samples. The theory of this method is explained, and it is demonstrated that relative viscosity and surface tension with contact angle can be obtained independently by measuring the capillary flow under differential pressure. We conducted capillary flow experiments with a small sample volume (less than 100 μl) using a measurement system comprising a flow measurement unit, capillary chip, and differential pressure mechanism. The relative viscosities of pure water and dilute ethanol solutions obtained in the experiment were in one-to-one correspondence with the values in the Chemical Handbook. This also shows that relative surface tension can be determined using these measurement results and the separately measured contact angle values.

Optimisation of the conditions of dispersive liquid–liquid microextraction for environmentally friendly determination of bisphenols and benzophenone in complex water matrices by LC-MS/MS

Due to well-known risks to human and environmental health, the increasing presence of several micropollutants even in very low concentrations in environmental matrices requires sustainable and advanced analytical methods to expand monitoring programs, particularly for water quality control. The objective of this study was to optimise the conditions of the dispersive liquid–liquid microextraction (DLLME) for a combined group of emerging micropollutants (bisphenol A, bisphenol S, bisphenol F, bisphenol AF and benzophenone) in complex water matrices. Ten variables were chosen as the most important for DLLME procedure and the Design of Experiments (DoE) approach was applied to find the most relevant variables and optimise the DLLME procedure. The optimised DLLME was then, applied to real complex water samples including raw and treated wastewaters and surface water in Rio de Janeiro city, Brazil, and the extracted samples were analysed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The variables vortex mixing time (VMT), Dispersive Solvent Volume (DSV) and Extractor Solvent Volume (ESV) are the most important among the variables evaluated. The recovery efficiencies ranged from 60 to 120% after optimisation the amounts of target compounds found in the five real aqueous matrices varied from 0.005 to 150 μg L-1. The association of optimised DLLME with the UPLC-MS/MS met the validation requirements adopted and generated analytical results of high quality. The advantages of this extraction technique include reduction of cost per analysis; low volume of samples and low volume of solvent required; no need to use cartridges; reduction of waste generation. The method is, therefore, suitable to process large numbers of samples, particularly in countries with limited budgets for environmental monitoring.

Cerium oxide-doped PEDOT nanocomposite for label-free electrochemical immunosensing of anti-p53 autoantibodies.

Alabel-free nanoimmunosensor is reportedbased on p53/CeO2/PEDOT nanobiocomposite-decorated screen-printed gold electrodes (SPAuE) for the electrochemical detection of anti-p53 autoantibodies. CeO2 nanoparticles (NPs) were synthesized and stabilized with cyanopropyltriethoxysilane by a soft chemistry method. The nanoimmunosensing architecture was prepared by in situ electropolymerization of 3,4-ethylenedioxythiophene (EDOT) on SPAuE in the presence of CeO2 NPs. The CeO2 NPs and Ce/PEDOT/SPAuE were characterized by scanning and transmission electron microscopy, dynamic and electrophoretic light scattering, ultraviolet-visible spectrophotometry, X-ray diffraction, Fourier-transform infrared spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. Ce/PEDOT/SPAuE was biofunctionalized with p53 antigen by covalent bonding for the label-free determination of anti-p53 autoantibodies by differential pulse voltammetry. The nanobiocomposite-based nanoimmunosensor detected anti-p53 autoantibodies in a linear range from 10 to 1000pgmL-1, with a limit of detection (LOD) of 3.2pgmL-1. The nanoimmunosensor offered high specificity, selectivity, and long-term storage stability with great potential to detect anti-p53 autoantibodies in serum samples. Overall, incorporating organo-functional nanoparticles into polymeric matrices can provide a simple-to-assemble, rapid, and ultrasensitive approach for on-site screening of anti-p53 autoantibodies and other disease-related biomarkers with low sample volumes.

Recent Advances in Aptasensors For Rapid and Sensitive Detection of Staphylococcus Aureus.

The infection of Staphylococcus aureus (S.aureus) and the spread of drug-resistant bacteria pose a serious threat to global public health. Therefore, timely, rapid and accurate detection of S. aureus is of great significance for food safety, environmental monitoring, clinical diagnosis and treatment, and prevention of drug-resistant bacteria dissemination. Traditional S. aureus detection methods such as culture identification, ELISA, PCR, MALDI-TOF-MS and sequencing, etc., have good sensitivity and specificity, but they are complex to operate, requiring professionals and expensive and complex machines. Therefore, it is still challenging to develop a fast, simple, low-cost, specific and sensitive S. aureus detection method. Recent studies have demonstrated that fast, specific, low-cost, low sample volume, automated, and portable aptasensors have been widely used for S. aureus detection and have been proposed as the most attractive alternatives to their traditional detection methods. In this review, recent advances of aptasensors based on different transducer (optical and electrochemical) for S. aureus detection have been discussed in details. Furthermore, the applications of aptasensors in point-of-care testing (POCT) have also been discussed. More and more aptasensors are combined with nanomaterials as efficient transducers and amplifiers, which appears to be the development trend in aptasensors. Finally, some significant challenges for the development and application of aptasensors are outlined.

Performance evaluation of the XT MicroSlide assay pairs on the Vitros XT 7600 compared to VITROS single microslide assays on Vitros 5600

ObjectivesPediatric hospitals are always challenged by specimen volumes and thus any innovation in this realm is very welcome. With the introduction of Microslide assay pairs, we aimed to evaluate the analytical performance of the Vitros XT MicroSlide assay pairs on the Vitros XT 7600 compared to single MicroSlides. DesignPerformance characteristics included within-run precision, analytical measurable range, method comparison, and interference verification. We compared six XT MicroSlide pairs on the Vitros XT 7600 with twelve corresponding single slide assays on the Vitros 5600 system. ResultsThe XT MicroSlides on Vitros XT 7600 demonstrated excellent precision, equivalent analytical measurable range, and strong method correlation with single slide assays on Vitros 5600 for most of the assays tested. Within-run CVs of the analytes ranged between 0.32% and 2.93% with between-run CV of less than 8.8% and linearity for all analytes was within the manufacturer's specified range. Interference studies showed comparable effects of hemolysis, lipemia, and bilirubin on both instruments. ConclusionsThe XT MicroSlides are comparable to the single MicroSlide assays with improved efficiency, turnaround times and lower sample volumes.

Quantifying up to 90 polyphenols simultaneously in human bio-fluids by LC-MS/MS

Establishing a method for human biomonitoring (HBM) of polyphenols enables the assessment of internal concentrations of these food bio-actives and the correlation with potential health effects such as antioxidant or anti-inflammatory properties. Thus, a targeted LC-MS/MS method for quantifying up to 90 analytes, representing the main polyphenol classes including flavanones, isoflavones, stilbenes, and phenolic acids, was developed for human urine, serum, and plasma. The method was established for low sample volumes and with a cost and time efficient sample preparation protocol for high-throughput, which is critical for its application in large cohort and exposome-wide association studies. On average, the sample preparation yielded extraction efficiencies of 98% for urine, 98% for serum, and 87% for plasma. Limits of detection were between 0.11 ng mL−1 and 300 ng mL−1 for urine, 0.12 ng mL−1 and 190 ng mL−1 for serum, and 0.12 ng mL−1 and 340 ng mL−1 for plasma, excluding one analyte. In-house validation revealed that 66, 49, and 64 analytes for urine, serum, and plasma, respectively, fulfilled all stringent requirements, that are usually utilized for tailored single analyte methods, at all evaluated concentration levels. After validation, this method was applied in a proof-of-principle study that detected 39 polyphenols in urine. Changes in the concentrations of the analytes after the ingestion of a high polyphenol smoothie was examined over 24 h. The study further confirmed that the majority of polyphenols detected were phenolic acids, and phase II conjugated metabolites were more abundant than their respective non-conjugated forms.

Electrochemically Effective Surface Area of a Polyaniline Nanowire-Based Platinum Microelectrode and Development of an Electrochemical DNA Sensor

Electrochemical DNA sensors based on nanocomposite materials of polyaniline nanowires (PANi NWs) have been published in the literature. However, it is interesting that there are very few research studies related to the development of electrochemical DNA sensors based on PANi NWs individually. In this study, PANi NWs were synthesized site-specifically on a Pt microelectrode with only 0.785 mm2 area using an electropolymerization procedure. The electrosynthesis allows direct deposition of PANi NWs onto the Pt microelectrode in a rapid and cost-effective way. The good properties of PANi NWs including uniform size, uniform distribution throughout the Pt working electrode, and H2SO4 doping which improved the conductivity of the PANi material were obtained. Especially, the electrochemically effective surface area of the PANi NW-based Pt microelectrode determined in this work is nearly 19 times larger than that of the Pt working electrode. The PANi NW layer with large electrochemically effective surface area and high biocompatibility is consistent with the application in electrochemical DNA sensors. The fabricated DNA sensors show advantages such as simple fabrication, direct detection, high sensitivity (with the detection limit of 2.48 × 10−14 M), good specificity, and low sample volume requirement. This study also contributes to confirm the role of PANi NWs in DNA probe immobilization as well as in electrochemical signal transmission in the development of electrochemical DNA sensors.