Small Volumes Of Serum Research Articles

Exosomal Micro RNA Isolation in White-Tailed Deer (Odocoileus virginianus) for Diagnostic Biomarker Discovery.

Molecular approaches are becoming more prevalent for the diagnosis of neurodegenerative diseases in human medicine and can be extended to diagnosis of wildlife diseases such as chronic wasting disease and other prion diseases. These diseases have been associated with exosome-bound molecular biomarkers of disease progression, such as proteins and micro RNA molecules (miRNA). We tested and optimized a method for exosomal miRNA isolation from minimally invasive, small-volume serum samples obtained from white-tailed deer (Odocoileus virginianus). We confirmed the isolation of exosomes and optimized a commercially available benchtop kit to obtain sufficient and pure RNA for miRNA sequencing. The selected method for RNA extraction combines two 500-μL serum aliquots into one elution column and re-eluting the final product of the column. We identified 137 miRNA present in healthy white-tailed deer that can be used as a baseline to identify putative miRNA biomarkers of disease progression and mechanisms of infection in future comparative disease studies. This approach to biomarker discovery may help to inform biological processes in wildlife populations and provide alternatives to invasive or postmortem samples.

Advanced Nanoencapsulation-Enabled Ultrasensitive Analysis: Unraveling Tumor Extracellular Vesicle Subpopulations for Differential Diagnosis of Hepatocellular Carcinoma via DNA Cascade Reactions.

Tumor-derived extracellular vesicles (tEVs) hold immense promise as potential biomarkers for the precise diagnosis of hepatocellular carcinoma (HCC). However, their clinical translation is hampered by their inherent characteristics, such as small size and high heterogeneity and complex environment, including non-EV particles and normal cell-derived EVs, which prolong separation procedures and compromise detection accuracy. In this study, we devised a DNA cascade reaction-triggered individual EV nanoencapsulation (DCR-IEVN) strategy to achieve the ultrasensitive and specific detection of tEV subpopulations via routine flow cytometry in a one-pot, one-step fashion. DCR-IEVN enables the direct and selective packaging of multiple tEV subpopulations in clinical serum samples into flower-like particles exceeding 600 nm. This approach bypasses the need for EV isolation, effectively reducing interference from non-EV particles and nontumor EVs. Compared with conventional analytical technologies, DCR-IEVN exhibits superior efficacy in diagnosing HCC owing to its high selectivity for tEVs. Integration of machine learning algorithms with DCR-IEVN resulted in differential diagnosis accuracy of 96.7% for the training cohort (n = 120) and 93.3% for the validation cohort (n = 30), effectively distinguishing HCC, cirrhosis, and healthy donors. This strategy offers a streamlined workflow and rapid assay completion and requires only small-volume serum samples and routine clinical devices, facilitating the clinical translation of tEV-based tumor diagnosis.

Validation of a Method Scope Extension for Simple Biomonitoring of 353 Pollutants in Serum Samples

Animals and humans are exposed to various residues that can have a detrimental impact on health, including carcinogenic potential, endocrine disruption, or fatal toxicity. The toxic burden can be evaluated in several biological samples, with serum being one of the preferred and most convenient options. In this study, we have applied and validated a method for detecting several hundred toxins in serum samples. This technique involved a single-step QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction followed by analysis using gas and liquid chromatography coupled with mass spectrometry. With this methodology, we could detect and quantify up to 353 compounds, including persistent organic pollutants (POPs), pesticides, pharmaceuticals, and rodenticides, using just 250 µL of serum. Among them, 92% could be measured at concentrations below 1.25 ng/mL, making it ideal for biomonitoring. We applied this method to samples collected from camels (n = 40) and humans (n = 25). We detected naproxen, ketoprofen, paracetamol, levamisole, and some POPs in these samples. This study validated the ability to simultaneously detect a broad range of compounds in small volumes of serum.

Nanoplasmonic immunosensor for the detection of SCG2, a candidate serum biomarker for the early diagnosis of neurodevelopmental disorder

The neural circuits of the infant brain are rapidly established near 6 months of age, but neurodevelopmental disorders can be diagnosed only at the age of 2–3 years using existing diagnostic methods. Early diagnosis is very important to alleviate life-long disability in patients through appropriate early intervention, and it is imperative to develop new diagnostic methods for early detection of neurodevelopmental disorders. We examined the serum level of secretogranin II (SCG2) in pediatric patients to evaluate its potential role as a biomarker for neurodevelopmental disorders. A plasmonic immunosensor performing an enzyme-linked immunosorbent assay (ELISA) on a gold nanodot array was developed to detect SCG2 in small volumes of serum. This nanoplasmonic immunosensor combined with tyramide signal amplification was highly sensitive to detect SCG2 in only 5 μL serum samples. The analysis using the nanoplasmonic immunosensor revealed higher serum SCG2 levels in pediatric patients with developmental delay than in the control group. Overexpression or knockdown of SCG2 in hippocampal neurons significantly attenuated dendritic arborization and synaptic formation. These results suggest that dysregulated SCG2 expression impairs neural development. In conclusion, we developed a highly sensitive nanoplasmonic immunosensor to detect serum SCG2, a candidate biomarker for the early diagnosis of neurodevelopmental disorders.

Alternative Method for HDL and Exosome Isolation with Small Serum Volumes and Their Characterizations

High-density lipoprotein (HDL) and exosomes are promising sources of biomarkers. However, the limited sample volume and access to the ultracentrifuge equipment are still an issue during HDL and exosome isolation. This study aimed to isolate HDL and exosomes using an ultracentrifugation-free method with various small serum volumes. HDL was isolated from 200 µL (HDL200) and 500 µL (HDL500) of sera. Three different volumes: 50 µL (Exo50), 100 µL (Exo100), and 250 µL (Exo250) were used for exosome isolation. HDL and exosomes were isolated using commercial kits with the modified method and characterized by multiple approaches. The HDL levels of HDL200 and HDL500 were not significantly different (p > 0.05), with percent recoveries of >90%. HDL200 and HDL500 had the same protein pattern with a biochemical similarity of 99.60 ± 0.10%. The particle sizes of Exo50, Exo100, and Exo250 were in the expected range. All isolated exosomes exhibited a similar protein pattern with a biochemical similarity of >99%. In conclusion, two different serum volumes (200 and 500 µL) and three different serum volumes (50, 100, and 250 µL) can be employed for HDL and exosome isolation, respectively. The possibility of HDL and exosome isolation with small volumes will accelerate biomarker discoveries with various molecular diagnostic approaches.

Induction of Ascites in Mice.

Smaller animals such as rats, mice, hamsters, and guinea pigs are usually poor choices for polyclonal antibody production because only small volumes of serum can be obtained. This problem can be reduced by inducing the formation of ascites in mice, which can provide up to 10 mL of ascites fluid from a single animal. Antibody titers in ascites fluids are almost as high as serum titers.

Detection and quantification of Mycobacterium tuberculosis antigen CFP10 in serum and urine for the rapid diagnosis of active tuberculosis disease

Outside of the ongoing COVID-19 pandemic, tuberculosis is the leading cause of infectious disease mortality globally. Currently, there is no commercially available point-of-care diagnostic that is rapid, inexpensive, and highly sensitive for the diagnosis of active tuberculosis disease. Here we describe the development and optimization of a novel, highly sensitive prototype bioelectronic tuberculosis antigen (BETA) assay to detect tuberculosis-specific antigen, CFP10, in small-volume serum and urine samples. In this proof-of-concept study we evaluated the performance of the BETA assay using clinical specimens collected from presumptive tuberculosis patients from three independent cohorts. Circulating CFP10 antigen was detected in ALL serum (n = 19) and urine (n = 3) samples from bacteriologically confirmed tuberculosis patients who were untreated or had less than one week of treatment at time of serum collection, successfully identifying all culture positive tuberculosis patients. No CFP10 antigen was detected in serum (n = 7) or urine (n = 6) samples from individuals who were determined to be negative for tuberculosis disease. Additionally, antigen quantification using the BETA assay of paired serum samples collected from tuberculosis patients (n = 8) both before and after treatment initiation, indicate consistently declining within-person levels of CFP10 antigen during treatment. This novel, low-cost assay demonstrates potential as a rapid, non-sputum-based, point-of-care tool for the diagnosis of tuberculosis disease.

Simultaneous Analyses of 5 Omega Fatty Acids on Dried Serum Spot using Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

Omega fatty acids play an important role as biomarkers of chronic inflammatory diseases such as asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, and inflammatory bowel disease. The purpose of our study was to develop a new analytical method for the detection of omega fatty acids from dried serum spots. We developed an ultrahigh performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS)-based method in the multiple reaction monitoring (MRM) mode with negative ionization. The detected MRM transitions for five omega fatty acids are as follow: α-linolenic acid (m/z=277.20→277.20), eicosapentaenoic acid (m/z=301.10→257.30), docosahexaenoic acid (m/z=327.00→283.15), arachidonic acid (m/z=303.20→259.25), and docosapentaenoic acid (m/z= 329.10→285.25). The calibration curve showed an excellent linearity within 0.1-2.5 μg/mL range (R2=0.9947~0.9994). The analytical methods howed excellent sensitivity (LOD: 0.005-0.01 μg/mL, LOQ: 0.03-0.1 μg/mL), which would allow for the targeted analyses of omega fatty acids. The recoveries of the omega fatty acid from the dried serum spots were 92.6%- 110.4% (RSD: ±0.2%-9.6%) and the retention time was within 5 min. This improved method offers rapid and sensitive quantification of omega fatty acids on dried serum spots using a small volume of serum and is expected to be applied to various biological specimens.

Simultaneous Measurement of 67 Non-Human Primate Immunological, Metabolic and Hormonal Biomarkers using Luminex® xMAP® Technology

Abstract Immunology, metabolic, and pituitary biomarker research in non-human primates (NHP) is essential for the study of several diseases and biological regulation. To meet this need, we developed three NHP MILLIPLEX® multiplex panels: 12-plex NHP Metabolic Hormone, 7-plex NHP Pituitary Panel 1, and our new 48-plex NHP Cytokine/Chemokine Panel A for the simultaneous measurement of picogram levels of 67 immune, metabolic and hormone factors, in small volumes of serum, plasma or cell culture samples using Luminex® xMAP® technology. Serum from in vivo LPS-challenged animals (n=2 cynomolgus, n=2 rhesus) demonstrated increased levels of 19 cytokines/chemokines with peak elevated levels at 1 hr for 4 analytes (e.g. IL-10, TNFα), at 3 hr for 11 analytes (e.g. IL-6, IFNγ) and at 8 hr (e.g. Fractalkine, I-TAC). Metabolic markers, GIP and GLP-1 (active) in rhesus, also increased significantly at 8 hr and 3 hr, respectively (p<0.02). Significant differences between fasted and fed serum sample values (n=5 cynomolgus, n=5 rhesus) were found for several metabolic markers, including C-peptide, GIP, Insulin and PP, which increased with feeding (P<0.05). In cynomolgus, I-TAC and IP-10 significantly increased after feeding (P<0.003). Testing healthy serum samples from three additional NHP species (African green monkey (n=5), baboon (n=5), chimpanzee (n=5)) exhibited overall sample detectability of 89%, 77%, and 64% for the MILLIPLEX®Cytokine/Chemokine Panel A, Pituitary, and Metabolic multiplex panels, respectively. Thus, the MILLIPLEX®NHP Metabolic Hormone, NHP Pituitary Panel 1 and our latest and largest NHP panel, the 48-plex NHP Cytokine/Chemokine Panel A, are valuable tools for profiling 67 biomarkers in 5 NHP species for disease research.

Microarray-Based Allergy Diagnosis: Quo Vadis?

More than 30% of the world population suffers from allergy. Allergic individuals are characterized by the production of immunoglobulin E (IgE) antibodies against innocuous environmental allergens. Upon allergen recognition IgE mediates allergen-specific immediate and late-phase allergic inflammation in different organs. The identification of the disease-causing allergens by demonstrating the presence of allergen-specific IgE is the key to precision medicine in allergy because it allows tailoring different forms of prevention and treatment according to the sensitization profiles of individual allergic patients. More than 30 years ago molecular cloning started to accelerate the identification of the disease-causing allergen molecules and enabled their production as recombinant molecules. Based on recombinant allergen molecules, molecular allergy diagnosis was introduced into clinical practice and allowed dissecting the molecular sensitization profiles of allergic patients. In 2002 it was demonstrated that microarray technology allows assembling large numbers of allergen molecules on chips for the rapid serological testing of IgE sensitizations with small volumes of serum. Since then microarrayed allergens have revolutionized research and diagnosis in allergy, but several unmet needs remain. Here we show that detection of IgE- and IgG-reactivity to a panel of respiratory allergens microarrayed onto silicon elements is more sensitive than glass-based chips. We discuss the advantages of silicon-based allergen microarrays and how this technology will allow addressing hitherto unmet needs in microarray-based allergy diagnosis. Importantly, it described how the assembly of silicon microarray elements may create different microarray formats for suiting different diagnostic applications such as quick testing of single patients, medium scale testing and fully automated large scale testing.

Personalized immunoglobulin aptamers for detection of multiple myeloma minimal residual disease in serum

Multiple myeloma (MM) is a neoplasm of plasma cells that secrete patient specific monoclonal immunoglobulins. A recognized problem in MM treatment is the early recognition of minimal residual disease (MRD), the major cause of relapse. Current MRD detection methods (multiparameter flow cytometry and next generation sequencing) are based on the analysis of bone marrow plasma cells. Both methods cannot detect extramedullary disease and are unsuitable for serial measurements. We describe the methodology to generate high affinity DNA aptamers that are specific to a patient’s monoclonal Fab region. Such aptamers are 2000-fold more sensitive than immunofixation electrophoresis and enabled detection and quantification of MRD in serum when conventional MRD methods assessed complete remission. The aptamer isolation process that requires small volumes of serum is automatable, and Fab specific aptamers are adaptable to multiple diagnostic formats including point-of-care devices.

Point-of-care quantification of serum cellular fibronectin levels for stratification of ischemic stroke patients

The abundance of cellular fibronectin (c-Fn) for ischemic stroke patients and the narrow time-window (<4.5 h) for the decision to administer the thrombolytic treatment with recombinant tissue plasminogen activator (rtPA) are challenging for the development of a point-of-care (PoC) diagnostic platform. We report a case of stratification of ischemic stroke patients based on a magnetoresistive biosensor platform that quantifies the c-Fn levels in a small volume of serum, within the clinically relevant time-window. Our PoC platform uses different ratios of biofunctionalized magnetic nanoparticles (MNPs) as immunoassay labels to adjust the sensitivity within the clinically relevant ranges for c-Fn (1-4 μg/mL). After optimizing the detection range, resolution, and sensitivity, our device was able to stratify ischemic stroke patients who developed hemorrhagic transformation, the main side-effect of rtPA, from those (both non-treated and treated with rtPA) who did not.

Two rapid, accurate liquid chromatography tandem mass spectrometry methods for the quantification of seven uremic toxins: An application for describing their accumulation kinetic profile in a context of acute kidney injury

Acute kidney injury (AKI) is a frequent, serious complication in critically ill patients. Even if renal replacement therapy is rapidly initiated, AKI may lead to the acute accumulation of metabolic waste products called uremic toxins (UTs). Although the accumulation and effects of UTs have been extensively described in the setting of chronic kidney disease (CKD), few data are available for AKI. A rapid, sensitive, specific method with simple sample preparation is required to facilitate routine blood monitoring of UTs in a context of acute accumulation.We have developed and validated two fast liquid chromatography tandem mass spectrometry methods for the quantification of seven UTs in human serum. The first method (in negative ionization mode) enables the quantification of five UTs (hippuric acid (HA), indoxyl sulfate (IxS), para-cresyl sulfate (pCS), para-cresyl glucuronide (pCG), 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF)). The second method (in positive ionization mode) enables the quantification of two UTs (indole-3-acetic acid (IAA), and trimethylamine N-oxide (TMAO)). Sample preparation consisted of the deproteinization of a small volume of serum (50 µL). The run-times required to assay all the UTs in negative and positive ionization modes were only 2.5 and 2 min, respectively. In order to obtain a reliable, toxin-free matrix for the preparation of calibration standards and quality controls, serum was pretreated with activated charcoal. We used these methods to determine the time course of UT accumulation in eight patients who developed an AKI after cardiac surgery.The calibration curves ranged from 0.1 to 100 µg mL−1 for all the UTs (except for IAA: 0.5 to 100 µg mL−1), and the correlation coefficients were above 0.999 for all. The methods were reproducible, repeatable, and accurate, with all coefficients of variation and biases below 15%. The highest concentrations measured in patients with AKI were lower than those reported in CKD stages 4 and 5 but higher than those observed in patients with no impairment of renal function (particularly for IxS and pCS). Our results also highlighted low accumulation of the other toxins (IAA, HA, TMAO, pCG, and CMPF). The UT concentrations did not rise earlier than that of creatinine; although the return to baseline took longer than for creatinine for some compounds. Lastly, assessment of the time course of UT accumulation as a prognostic marker for AKI (particularly for pCS and IxS) appears to be promising and should be continued in a larger number of patients.

Improvement of the sensitivity of the detection of Gal d 6-specific IgE via biotinylation in vivo

Introduction and objectivesThis study aimed to compare the effects of different biotinylation methods on the performance characteristics of allergen-specific IgE detection. Materials and methodsThe Gal d 6 gene was cloned into the pAN6/pAC6 vector, resulting in rGal d 6-Bio/Bio-rGal d 6 vector. The fusion protein was expressed in Escherichia coli AVB101 and simultaneously biotinylated in a site-specific manner. The Gal d 6 gene was amplified via PCR and cloned into the pET-28a vector and transformed into E. coli BL21 and purified via Ni-NTA, followed by chemical biotinylation using Sulfo-NHS-LC-Biotin. Twenty-eight patients allergic to hen's egg white were examined for sensitization against egg yolk. An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed to detect allergen-specific IgE. ResultsrGal d 6, Bio-rGal d 6, and rGal d 6 were prepared using different biotin binding modes to detect allergen-specific IgE. rGal d 6-Bio (Kd=0.6154) and Bio-rGal d 6 (Kd=0.6698) had a markedly better detection performance than rGal d 6 (Kd=28.93), and the rGal d 6-Bio had a better detection performance in small-volume serum samples. ConclusionsrGal d 6-Bio improved the sensitivity for the detection of allergen-specific IgE.

Endocrine, Metabolic and Pharmacological Effects of Thyronamines (TAM), Thyroacetic Acids (TA) and Thyroid Hormone Metabolites (THM) - Evidence from in vitro, Cellular, Experimental Animal and Human Studies.

Thyroid hormone metabolites (THM) with few or no iodine substituents such as 3,5-T2, the thyronamines 3-T1AM and T0AM, and their oxidation products, the thyroacetic acids (TA) formed by monoamine oxidases, have recently attracted major interest due to their metabolic actions which are in part distinct from those of the classical thyromimetic hormone T3, the major ligand of T3 receptors. This review compiles and discusses in vitro effects of 3,5-T2, TAM and TA reported for thyrocytes, pancreatic islets and hepatocytes as well as findings from in vivo studies in mouse models after single or repeated administration of pharmacological doses of these agents. Comparison of the 3,5-T2 effects on the transcriptome with not yet published proteome data in livers of obese mice on high fat diet indicate a distinct anti-steatotic effect of this THM. Furthermore, uptake, metabolism, and cellular actions via various receptors such as trace amine-associated receptors (TAAR), alpha-adrenergic, GPCR and T3 receptors are discussed. Studies on postulated pathways of biosynthesis of 3-T1AM, its effects on the HPT-axis and thyroid gland as well as insulin secretion are reviewed. 3-T1AM also acts on hepatocytes and interferes with TRPM8-dependent signaling in human cell lines related to the eye compartment. Human studies are presented which address potential biosynthesis routes of 3,5-T2 and 3-T1AM from THM precursors, especially T3. The current state of diagnostic analytics of these minor THM in human blood is portrayed comparing and critically discussing the still divergent findings based on classical immunoassay and recently developed liquid-chromatography/mass- spectrometry methods, which allow quantification of the thyronome spectrum from one single small volume serum sample. The clinical perspectives of use and potential abuse of these biologically active THM is addressed.

Estimated PCDD/F TEQ and total TEQ concentrations in the serum of 7–10 year old Finnish children

Polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs) and polychlorinated biphenyls (PCBs) are persistent organic pollutants that have detrimental health effects. As people are exposed to them mainly through the diet, EU has set maximum food dioxin and PCBs levels. EFSA CONTAM Panel made new risk assessment in 2018 that lowered the tolerable weekly intake (TWI) from 14 pg-TEQ/kg bw/week to 2 pg-TEQ/kg bw/week. Critical effect was decreased semen count at the age of 18–19 years if serum total TEQ at the age of 9 years exceeded the No Observed Adverse Effect Level (NOAEL) of 7 pg/g lipid. However, it is largely unknown to what extent NOAEL is exceed in European boys currently. We thus measured PCBs from small volume of serum in 184 Finnish children 7–10 years of age. To estimate the TEQ levels of children from measured PCB levels, we used our existing human milk PCDD/F and PCB concentrations to create a hierarchical Bayesian regression model that was used to estimate TEQs from measured PCBs. For quality control (QC), three pooled blood samples from 18 to 20 year old males were measured for PCDD/Fs and PCBs, and estimated for TEQs. In QC samples measured and estimated TEQs agreed within 84%–106%. In our estimate for 7–10 year old children, PCDD/F TEQ exceeded NOAEL only in 0.5% and total TEQ in 2.7% of subjects. Risk management following the decreased TWI proposed by the CONTAM Panel should be carefully considered if total TEQ in children is already largely below the NOAEL.

Biological Evaluation of the MILLIPLEX® Human Cytokine/Chemokine/Growth Factor Panel A for Disease Profiling of 48 Immune Factors

Abstract Cytokine, chemokine and growth factor research plays a significant role in achieving a deeper understanding of the immune system and disease. We recently developed a MILLIPLEX® Human Cytokine/Chemokine/Growth Factor Panel A multiplex assay for simultaneous measurement of picogram levels of 48 human immune factors using Luminex® xMAP® technology in small volumes of serum, plasma or cell culture samples. Distinctly different stimulant-dependent cytokine responses were observed for challenged human PBMCs. Both LPS and Con-A induced similarly the secretion of 13 analytes, whereas LPS preferentially induced 10 analytes, including TNFα, IL-1α and IL-1β. Con-A preferentially induced 16 analytes including Th17 cell cytokines IL-17A and IL-17F. Plasma samples, from active (n=20) and benign (n=18) ovarian cancer patients (Dr. Richard Moore, U of Rochester Medical Center) were tested at UPMC. Fourteen analytes were significantly elevated in active patients vs. benign patients including: cytokines TNFα, IL-5, M-CSF; chemokines MCP-3, Fractalkine; Growth Factors FLT-3L, PDGF-BB (p&amp;lt;0.05); cytokines IL-6, IL-18 (p&amp;lt;0.02); cytokines IL-7, IL-27, IL-10, IL-13 (key role in cancer pathogenesis); chemokine MIP-1β (p&amp;lt;0.01). Residual serum and plasma sepsis patient samples (n=16) were compared to those from healthy controls (n=20) and 7 analytes were significantly elevated in sepsis patients: cytokines IL-1RA, IL-6 (p&amp;lt;0.05), IL-7, IL-27, MIP-1β (p&amp;lt;0.02), M-CSF; chemokine MIG (p&amp;lt;0.01). Twelve other analytes trended higher in sepsis samples. Thus, the Human Cytokine/Chemokine/Growth Factor Panel A is a useful tool for profiling 48 immune factors both in cell culture or blood samples for the study of human disease and inflammation.

Integrated Single Microbead-Arrayed μ-Fluidic Platform for the Automated Detection of Multiplexed Biomarkers.

An automated, single microbead-arrayed μ-fluidic immunoassay (AMIA) device is innovatively devised in this study, which enables the highly sensitive and simultaneous detection of multiplex biomarkers with fully automatic operations. The AMIA platform not only achieves automated assay processing and multiplexed target detection by integrating single microbead manipulation, sample loading, multistep washing, and immunoreaction on a microfluidic chip but also confers high sensitivity due to the highly efficient signal enriching effect on a single microbead by the use of only a routine sandwich immunoreaction. As such, as low as the pg/mL level of multiplexed protein biomarkers can be simultaneously determined in a quite small volume of serum (∼20 μL is enough), which can well meet the clinical demand for disease screening and prognosis. What is more, the detection results of several clinically important biomarkers in clinical samples with the AMIA platform exhibit excellent consistency with those obtained by using a standard clinical test. Thus, in virtue of the excellent features in terms of high sensitivity, multiplexing capability, generality, and high degree of automation, the AMIA provides a practical and user-friendly platform for assaying different biomarkers in clinical diagnostics and point-of-care testing.

A rapid, automatic and accurate assay for quantifying temocillin in human serum and CSF using turbulent flow liquid chromatography coupled to high-resolution mass spectrometry. Clinical application.

Temocillin is a β-lactamase-resistant penicillin used for the treatment of multiple drug-resistant Gram-negative bacteria. To maximize efficacy and avoid adverse effects, the dose regimen has to be quickly adjusted to the clinical situations. This necessitates the development of a rapid, reliable and accurate analytical method. Temocillin and the stable isotopically labeled internal standard ([13 C6 ]-amoxicillin) were extracted from either serum or cerebrospinal fluid by a turbulent flow liquid chromatographic method and eluted onto an octadecyl-silica phase with polar endcapping. Mass spectrometry was conducted using an exact mass determination method by electrospray positive ionization high-resolution mass spectrometry. The LLOQ and ULOQ of the present method were determined to be 0.4 and 200 μg/ml for serum and cerebrospinal fluid samples, respectively. The total analysis time was <7 min. The recovery ranged from 87.7 to 120.8%. Intra- and inter-day precision and trueness were tested at four concentration levels: 0.4, 8, 40 and 160 μg/ml. Values were 6.33 ± 1.53, 8.8 ± 1.3, 8.8 ± 0.36 and 2.1 ± 0.76%, and 5.0 ± 0.54, 9.9 ± 1.0, 5.8 ± 1.6 and 0.1 ± 1.1%, for inter- and intra-day analysis, respectively. Temocillin was found to be stable under all relevant laboratory conditions. The method was cross-validated with a microbiological assay. This method is suitable for accurate measurement of temocillin concentration in small volumes of serum or cerebrospinal fluid. Thanks to the online extraction procedure, the overall analytical time is compatible with high-throughput analysis for clinical application.

Ultra-Sensitive Automated Profiling of EpCAM Expression on Tumor-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are abundant in most biological fluids and considered promising biomarker candidates, but the development of EV biomarker assays is hindered, in part, by their requirement for prior EV purification and the lack of standardized and reproducible EV isolation methods. We now describe a far-field nanoplasmon-enhanced scattering (FF-nPES) assay for the isolation-free characterization of EVs present in small volumes of serum (< 5 µl). In this approach, EVs are captured with a cancer-selective antibody, hybridized with gold nanorods conjugated with an antibody to the EV surface protein CD9, and quantified by their ability to scatter light when analyzed using a fully automated dark-field microscope system. Our results indicate that FF-nPES performs similarly to EV ELISA, when analyzing EV surface expression of epithelial cell adhesion molecule (EpCAM), which has clinical significant as a cancer biomarker. Proof-of-concept FF-nPES data indicate that it can directly analyze EV EpCAM expression from serum samples to distinguish early stage pancreatic ductal adenocarcinoma patients from healthy subjects, detect the development of early stage tumors in a mouse model of spontaneous pancreatic cancer, and monitor tumor growth in patient derived xenograft mouse models of pancreatic cancer. FF-nPES thus appears to exhibit strong potential for the direct analysis of EV membrane biomarkers for disease diagnosis and treatment monitoring.