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A clever approach for biosensing is to leverage the concept of the proximity effect, where analyte binding to probes can be coupled to a second, controlled binding event such as short DNA strands. This analyte-dependent effect has been exploited in various sensors with optical or electrochemical readouts. Electrochemical proximity assays (ECPA) are more amenable to miniaturization and adaptation to the point-of-care, yet ECPA has been generally targeted toward protein sensing with antibody-oligonucleotide probes. Antibodies themselves are also important as biomarkers, since they are produced in bodily fluids in response to various diseases or infections, often in low amounts. In this work, by using antigen-DNA conjugates, we targeted an ECPA method for antibody sensing and showed that the assay performance can be greatly enhanced using flexible spacers in the DNA conjugates. After adding flexible polyethylene glycol (PEG) spacers at two distinct positions, the spacers ultimately increased the antibody-dependent current by a factor of 4.0 without significant background increases, similar to our recent work using thermofluorimetric analysis (TFA). The optimized ECPA was applied to anti-digoxigenin antibody quantification at concentrations ranging over two orders of magnitude, from the limit of detection of 300 pM up to 50 nM. The assay was functional in 90% human serum, where increased ionic strength was used to counteract double-layer repulsion effects at the electrode. This flexible-probe ECPA methodology should be useful for sensing other antibodies in the future with high sensitivity, and the mechanism for signal improvement with probe flexibility may be applicable to other DNA-based electrochemical sensor platforms. [ABSTRACT FROM AUTHOR]

Sphingolipids play crucial roles in cellular membranes, myelin stability, and signalling responses to physiological cues and stress. Among them, sphingosine 1-phosphate (S1P) has been recognized as a relevant biomarker for neurodegenerative diseases, and its analogue FTY-720 has been approved by the FDA for the treatment of relapsing–remitting multiple sclerosis. Focusing on these targets, we here report three novel polymeric capture phases for the selective extraction of the natural biomarker and its analogue drug. To enhance analytical performance, we employed different synthetic approaches using a cationic monomer and a hydrophobic copolymer of styrene-DVB. Results have demonstrated high affinity of the sorbents towards S1P and fingolimod phosphate (FTY-720-P, FP). This evidence proved that lipids containing phosphate diester moiety in their structures did not constitute obstacles for the interaction of phosphate monoester lipids when loaded into an SPE cartridge. Our suggested approach offers a valuable tool for developing efficient analytical procedures. [ABSTRACT FROM AUTHOR]

In this study, we developed a customized high-resolution mass spectrometry metabolomics workflow integrating the dual sugar test employing lactulose and mannitol as test probes for intestinal permeability assessment with untargeted screening of small molecules. Urine samples were collected from patients with major depression and healthy controls as part of a clinical study at the psychiatric department. Using a dual injection/dual chromatography setup, the test probes were quantified by hydrophilic interaction liquid chromatography (HILIC) in a targeted assay, while drugs and their metabolites were profiled in an untargeted manner by reversed-phase separation. Rigorous method development and validation allowed for selective separation of sugar isomers and consequently accurate quantification of lactulose and mannitol in urine. Internal standardization with compound specific stable isotope-labeled standards enabled excellent analytical figures of merit such as high recoveries, precision (< 5%), and working range (5 orders of magnitude). Within one analytical run, intestinal permeability was assessed together with drugs and their metabolites, allowing to screen for confounding drugs and patient compliance to the therapeutic scheme., Competing Interests: Declarations. Ethics approval: Ethical approval was granted by the ethics committee of the Medical University of Vienna (EK-Nr: 1882/2020). Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The early detection of head and neck cancer is a prolonged challenging task. It requires a precise and accurate identification of tissue alterations as well as a distinct discrimination of cancerous from healthy tissue areas. A novel approach for this purpose uses microspectroscopic techniques with special focus on hyperspectral imaging (HSI) methods. Our proof-of-principle study presents the implementation and application of darkfield elastic light scattering spectroscopy (DF ELSS) as a non-destructive, high-resolution, and fast imaging modality to distinguish lingual healthy from altered tissue regions in a mouse model. The main aspect of our study deals with the comparison of two varying HSI detection principles, which are a point-by-point and line scanning imaging, and whether one might be more appropriate in differentiating several tissue types. Statistical models are formed by deploying a principal component analysis (PCA) with the Bayesian discriminant analysis (DA) on the elastic light scattering (ELS) spectra. Overall accuracy, sensitivity, and precision values of 98% are achieved for both models whereas the overall specificity results in 99%. An additional classification of model-unknown ELS spectra is performed. The predictions are verified with histopathological evaluations of identical HE-stained tissue areas to prove the model's capability of tissue distinction. In the context of our proof-of-principle study, we assess the Pushbroom PCA-DA model to be more suitable for tissue type differentiations and thus tissue classification. In addition to the HE-examination in head and neck cancer diagnosis, the usage of HSI-based statistical models might be conceivable in a daily clinical routine. [ABSTRACT FROM AUTHOR]

While many clinical laboratory tests are now highly automated, body fluid cell counting, particularly in low-cellularity samples such as cerebral spinal fluid (CSF), is often performed manually. Here, we report a simple, cost-effective method to obtain white and red blood cell counts from human body fluids such as CSF. The method consists of a compact, automated, and low-cost fluorescence microscope system, coupled to a sample chamber containing all of the necessary reagents in dry form to stain and prepare the sample. Sample focus and scanning are handled automatically, and the acquired multimodal images are automatically analyzed to extract cell counts. Comparison with manual counting on over 200 clinical samples shows excellent agreement. As the system counts a substantially larger image region than a standard manual cell count, we find our sensitivity to extremely low cellularity samples to potentially be higher than the manual gold standard, evidenced by our system recording images of cells in samples whose cell count was registered as "0" by a trained user. Thus, our system holds promise for routine, automated, and sensitive analysis of body fluids whose cellularity extends across a wide dynamic range. [ABSTRACT FROM AUTHOR]

This work presents a method to unequivocally detect urine sample tampering in cases where integrity of the sample needs to be verified prior to urinalysis. The technique involves the detection of distinct patterns of a triplex short tandem repeats system in DNA extracted from human urine. The analysis is realized with single-dye fluorescence detection and using a regular smartphone camera. The experimental results had demonstrated the efficacy of the analytical approach to obtaining distinct profiles of amplicons in urine from different sample providers. Reproducibility tests with fresh and stored urine have revealed a maximum variation in the profiles within an interval of 5 to 9%. Cases of urine sample tampering via mixture were simulated in the study, and the experiments have identified patterns of mixed genotypes from dual mixtures of urine samples. Moreover, sample adulteration by mixing a non-human fluid with urine in a volume ratio over 25% can be detected. The low cost of the approach is accompanied by the compatibility of the technique to use with different DNA sample preparation protocols and PCR instrumentation. Furthermore, the possibility of realizing the method in an integrated microchip system open great perspectives to conducting sample integrity tests at the site of urine sample reception and/or at resource-limited settings. [ABSTRACT FROM AUTHOR]

We have developed multichannel integrated microfluidic devices for automated preconcentration, labeling, purification, and separation of preterm birth (PTB) biomarkers. We fabricated multilayer poly(dimethylsiloxane)-cyclic olefin copolymer (PDMS-COC) devices that perform solid-phase extraction (SPE) and microchip electrophoresis (μCE) for automated PTB biomarker analysis. The PDMS control layer had a peristaltic pump and pneumatic valves for flow control, while the PDMS fluidic layer had five input reservoirs connected to microchannels and a μCE system. The COC layers had a reversed-phase octyl methacrylate porous polymer monolith for SPE and fluorescent labeling of PTB biomarkers. We determined μCE conditions for two PTB biomarkers, ferritin (Fer) and corticotropin-releasing factor (CRF). We used these integrated microfluidic devices to preconcentrate and purify off-chip-labeled Fer and CRF in an automated fashion. Finally, we performed a fully automated on-chip analysis of unlabeled PTB biomarkers, involving SPE, labeling, and μCE separation with 1 h total analysis time. These integrated systems have strong potential to be combined with upstream immunoaffinity extraction, offering a compact sample-to-answer biomarker analysis platform. [ABSTRACT FROM AUTHOR]

Today’s high-resolution mass spectrometers (HRMS) allow bioanalysts to perform untargeted/global determinations that can reveal unexpected compounds or concentrations in a patient’s sample. This could be performed for preliminary diagnosis attempts when usual diagnostic processes and targeted determinations fail. We have evaluated an untargeted diagnostic screening (UDS) procedure. UDS is a metabolome analysis that compares one sample (e.g., a patient) with control samples (a healthy population). Using liquid chromatography (LC)-HRMS full-scan analysis of human serum extracts and unsupervised data treatment, we have compared individual samples that were spiked with one xenobiotic or a higher level of one endogenous compound with control samples. After the use of different filters that drastically reduced the number of metabolites detected, the spiked compound was eventually revealed in each test sample and ranked. The proposed UDS procedure appears feasible and reliable to reveal unexpected xenobiotics (toxicology) or higher concentrations of endogenous metabolites. HRMS-based untargeted approaches could be useful as preliminary diagnostic screening when canonical processes do not reveal disease etiology nor establish a clear diagnosis and could reduce misdiagnosis. On the other hand, the risk of overdiagnosis of this approach should be reduced with mandatory biomedical interpretation of the patient’s UDS results and with confirmatory targeted and quantitative determinations.

The present study describes a development of nanohydrogel, loaded with QD and manganese (QD@Nanogel and QD@Mn@Nanogel), and its passive and electro-assisted delivery in solid tumors, visualized by fluorescence imaging and magnetic resonance imaging (MRI) on colon cancer-grafted mice as a model. QD@Nanogel was delivered passively predominantly into the tumor, which was visualized in vivo and ex vivo using fluorescent imaging. The fluorescence intensity increased gradually within 30 min after injection, reached a plateau between 30 min and 2 h, and decreased gradually to the baseline within 24 h. The fluorescence intensity in the tumor area was about 2.5 times higher than the background fluorescence. A very weak fluorescent signal was detected in the liver area, but not in the areas of the kidneys or bladder. This result was in contrast with our previous study, indicating that FITC@Mn@Nanogel did not enter into the tumor and was detected rapidly in the kidney and bladder after i.v. injection [J. Mater. Chem. B 2013, 1, 4932-4938]. We found that the embedding of a hard material (as QD) in nanohydrogel changes the physical properties of the soft material (decreases the size and negative charge and changes the shape) and alters its pharmacodynamics. Electroporation facilitated the delivery of the nanohydrogel in the tumor tissue, visualized by fluorescent imaging and MRI. Strong signal intensity was recorded in the tumor area shortly after the combined treatment (QD@Mn@Nanogel + electroporation), and it was observed even 48 h after the electroporation. The data demonstrate more effective penetration of the nanoparticles in the tumor due to the increased permeability of blood vessels at the electroporated area. There was no rupture of blood vessels after electroporation, and there were no artifacts in the images due to a bleeding. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Hip replacements are used to improve the quality of life of people with orthopaedic conditions, but the use of metal-on-metal (MoM) arthroplasty has led to poor outcomes for some patients. These problems are related to the generation of micro- to nanosized metal wear particles containing Cr, Co or other elements, but the current analytical methods used to investigate the processes involved do not provide sufficient information to understand the size or composition of the wear particles generated in vivo. In this qualitative feasibility study, asymmetric flow field-flow fractionation (AF) coupled with inductively coupled plasma mass spectrometry (ICP-MS) was used to investigate metal protein binding and the size and composition of wear metal particles present in serum and hip aspirates from MoM hip replacement patients. A well-established HPLC anion exchange chromatography (AEC) separation system coupled to ICP-MS was used to confirm the metal -protein associations in the serum samples. Off-line single particle ICP-MS (spICP-MS) analysis was used to confirm the approximate size distribution indicated by AF of the wear particles in hip aspirates. In the serum samples, AF -ICP-MS suggested that Cr was associated with transferrin (Tf) and Co with albumin (Alb) and an unidentified species; AEC -ICP-MS confirmed these associations and also indicated an association of Cr with Alb. In the hip aspirate sample, AF-ICP-MS suggested that Cr was associated with Alb and Tf and that Co was associated with Alb and two unidentified compounds; AEC analysis confirmed the Cr results and the association of Co with Alb and a second compound. Enzymatic digestion of the hip aspirate sample, followed by separation using AF with detection by UV absorption (280 nm), multi-angle light scattering and ICP-MS, suggested that the sizes of the Cr-, Co- and Mo-containing wear particles in a hip aspirate sample were in the range 40-150 nm. Off-line spICP-MS was used to confirm these findings for the Co- and Cr-containing nanoparticles. Whilst limited in scope, the results are sufficient to show the interaction of ions with transport proteins and give an indication of particle size, providing useful pathological indices. As such, the methods indicate a new way forward for in vivo investigation of the processes which lead to tissue necrosis and hip loosening in patients with MoM hip replacements. [ABSTRACT FROM AUTHOR]

The present work describes the development and optimization of electrochemical biosensors for specific determination of the biogenic polyamine spermine (Spm) and spermidine (Spmd) whose assessment represents a novel important analytical tool in food analysis and human diagnostics. These biosensors have been prepared using novel engineered enzymes: polyamine oxidase (PAO) endowed with selectivity towards Spm and Spmd and spermine oxidase (SMO) characterized by strict specificity towards Spm. The current design entails biosensors in which the enzymes were entrapped in poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ), a photocrosslinkable gel, onto an electrode surface. Screen-printed electrodes (SPEs) were used as electrochemical transducers for enzymatically produced hydrogen peroxide, operating at different potential vs Ag/AgCl according to the material of the working electrode (WE): +700 mV for graphite (GP) or −100 mV for Prussian blue (PB)-modified SPE, respectively. Biosensor performances were evaluated by means of flow injection amperometric (FIA) measurements. The modified electrodes showed good sensitivity, long-term stability and reproducibility. Under optimal conditions, the PAO biosensor showed a linear range 0.003-0.3 mM for Spm and 0.01-0.4 mM for Spmd, while with the SMO biosensor, a linear range of 0.004-0.5 mM for Spm has been obtained. The main kinetic parameters apparent Michaelis constant ( K), turnover number ( K) and steady-state current ( I) were determined. The proposed device was then applied to the determination of biogenic amines in blood samples. The results obtained were in good agreement with those obtained with the GC-MS reference method. [ABSTRACT FROM AUTHOR]

A critical review is presented on the use of linear and nonlinear Raman microspectroscopy in biomedical diagnostics of bacteria, cells, and tissues. This contribution is combined with an overview of the achievements of our research group. Linear Raman spectroscopy offers a wealth of chemical and molecular information. Its routine clinical application poses a challenge due to relatively weak signal intensities and confounding overlapping effects. Nonlinear variants of Raman spectroscopy such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) have been recognized as tools for rapid image acquisition. Imaging applications benefit from the fact that contrast is based on the chemical composition and molecular structures in a label-free and nondestructive manner. Although not label-free, surface enhanced Raman scattering (SERS) has also been recognized as a complementary biomedical tool to increase sensitivity. The current state of the art is evaluated, illustrative examples are given, future developments are pointed out, and important reviews and references from the current literature are selected. The topics are identification of bacteria and single cells, imaging of single cells, Raman activated cell sorting, diagnosis of tissue sections, fiber optic Raman spectroscopy, and progress in coherent Raman scattering in tissue diagnosis. The roles of networks-such as Raman4clinics and CLIRSPEC on a European level-and early adopters in the translation, dissemination, and validation of new methods are discussed. [ABSTRACT FROM AUTHOR]

Circulating cell-free DNA (cfDNA) is becoming an important clinical analyte for prenatal testing, cancer diagnosis and cancer monitoring. The extraction stage is critical in ensuring clinical sensitivity of analytical methods measuring minority nucleic acid fractions, such as foetal-derived sequences in predominantly maternal cfDNA. Consequently, quality controls are required for measurement of extraction efficiency, fragment size bias and yield for validation of cfDNA methods. We evaluated the utility of an external DNA spike for monitoring these parameters in a study comparing three specific cfDNA extraction methods [QIAamp® circulating nucleic acid (CNA) kit, NucleoSpin® Plasma XS (NS) kit and FitAmp™ plasma/serum DNA isolation (FA) kit] with the commonly used QIAamp DNA blood mini (DBM) kit. We found that the extraction efficiencies of the kits ranked in the order CNA kit > DBM kit > NS kit > FA kit, and the CNA and NS kits gave a better representation of smaller DNA fragments in the extract than the DBM kit. We investigated means of improved reporting of cfDNA yield by comparing quantitative PCR measurements of seven different reference gene assays in plasma samples and validating these with digital PCR. We noted that the cfDNA quantities based on measurement of some target genes (e.g. TERT) were, on average, more than twofold higher than those of other assays (e.g. ERV3). We conclude that analysis and averaging of multiple reference genes using a GeNorm approach gives a more reliable estimate of total cfDNA quantity. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Although dried blood spot (DBS) sampling is increasingly receiving interest as a potential alternative to traditional blood sampling, the impact of hematocrit (Hct) on DBS results is limiting its final breakthrough in routine bioanalysis. To predict the Hct of a given DBS, potassium (K) proved to be a reliable marker. The aim of this study was to evaluate whether application of an algorithm, based upon predicted Hct or K concentrations as such, allowed correction for the Hct bias. Using validated LC-MS/MS methods, caffeine, chosen as a model compound, was determined in whole blood and corresponding DBS samples with a broad Hct range (0.18-0.47). A reference subset ( n = 50) was used to generate an algorithm based on K concentrations in DBS. Application of the developed algorithm on an independent test set ( n = 50) alleviated the assay bias, especially at lower Hct values. Before correction, differences between DBS and whole blood concentrations ranged from −29.1 to 21.1 %. The mean difference, as obtained by Bland-Altman comparison, was −6.6 % (95 % confidence interval (CI), −9.7 to −3.4 %). After application of the algorithm, differences between corrected and whole blood concentrations lay between −19.9 and 13.9 % with a mean difference of −2.1 % (95 % CI, −4.5 to 0.3 %). The same algorithm was applied to a separate compound, paraxanthine, which was determined in 103 samples (Hct range, 0.17-0.47), yielding similar results. In conclusion, a K-based algorithm allows correction for the Hct bias in the quantitative analysis of caffeine and its metabolite paraxanthine. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: A simple and sensitive new method for the determination of d-3-hydroxybutyric acid (D-3-HBA) in human plasma after derivatization is described. The proposed method is based on the reaction of (2S)-2-amino-3-methyl-1-[4-(7-nitro-benzo-2,1,3-oxadiazol-4-yl)-piperazin-1-yl]-butan-1-one (NBD-PZ-Val) with D-3-HBA in the presence of O-(7-azobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and N-ethyldiisopropylamine (DIEA) to produce a fluorescent derivative. The formed derivative was monitored fluorimetrically at λex =489nm and λem =532nm. The HPLC analysis was carried out by use of a C18 analytical column (Synergy Hydro 150mm×3mm, i.d., 4μm) with a binary gradient elution program of 0.1% aqueous trifluoroacetic acid versus methanol. The method showed satisfactory linearity (r2 =0.9997) in the range from 20 to 500μmol/L. The limit of detection (LOD) of the method was 7.7μmol/L, while the limit of quantitation (LOQ) was 25.8μmol/L. The analytical method was successfully applied to human plasma samples from normal healthy subjects. [Copyright &y& Elsevier]

Molecular analytical methods are increasingly needed for a quick and reliable analysis of tissue in an operating room to provide more information during operations. In this Trends article, we highlight the current state and the developments of optical spectroscopic methods as intra operative tools. The clinical problem and challenges are illustrated on the example of brain tumor surgery. While fluorescence microscopy is already used, vibrational spectroscopy techniques will complement the standard method for brain tissue diagnostics. New portable instruments are currently available and can be stationed in the operating room for quick evaluation of tissue. The promise and limitations of fluorescence and vibrational spectroscopy as intraoperative tools are surveyed in this report. [ABSTRACT FROM AUTHOR]

The use of the amount of certain proteins in biological samples as markers for distinguishing between a healthy and a diseased state has become increasingly important in clinical diagnosis. As about 30 % of all proteins contain metals in one form or another, either as a cofactor or covalently bound as part of the protein, some of these proteins are regularly analyzed in clinical laboratories. With the increasing number of measurements of those proteins performed all over the world, the necessity of obtaining reliable and comparable results is becoming a focal point for scientists and politicians. Directives such as the EC directive covering in vitro diagnostic medical devices (Directive 98/79/EC) and standards such as EN ISO 17511:2003 demand the traceability of the results obtained for analytes in samples of human origin. However, no reference measurement procedures with results traceable to the SI exist for many metalloproteins. In this article, the situation for a few important metalloproteins, such as hemoglobin, transferrin, superoxide dismutase, ceruloplasmin, and C-reactive protein, for which specific efforts have been made in recent years to achieve comparable and traceable results worldwide, is discussed. These proteins also serve as examples of the difficulties scientists face when they wish to quantify proteins and the pitfalls they should avoid to achieve reliable results. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Needle trap devices (NTDs) have shown many advantages such as improved detection limits, reduced sampling time and volume, improved stability, and reproducibility if compared with other techniques used in breath analysis such as solid-phase extraction and solid-phase micro-extraction. Effects of sampling flow (2-30 ml/min) and volume (10-100 ml) were investigated in dry gas standards containing hydrocarbons, aldehydes, and aromatic compounds and in humid breath samples. NTDs contained (single-bed) polymer packing and (triple-bed) combinations of divinylbenzene/Carbopack X/Carboxen 1000. Substances were desorbed from the NTDs by means of thermal expansion and analyzed by gas chromatography-mass spectrometry. An automated CO-controlled sampling device for direct alveolar sampling at the point-of-care was developed and tested in pilot experiments. Adsorption efficiency for small volatile organic compounds decreased and breakthrough increased when sampling was done with polymer needles from a water-saturated matrix (breath) instead from dry gas. Humidity did not affect analysis with triple-bed NTDs. These NTDs showed only small dependencies on sampling flow and low breakthrough from 1-5 %. The new sampling device was able to control crucial parameters such as sampling flow and volume. With triple-bed NTDs, substance amounts increased linearly with increasing sample volume when alveolar breath was pre-concentrated automatically. When compared with manual sampling, automatic sampling showed comparable or better results. Thorough control of sampling and adequate choice of adsorption material is mandatory for application of needle trap micro-extraction in vivo. The new CO-controlled sampling device allows direct alveolar sampling at the point-of-care without the need of any additional sampling, storage, or pre-concentration steps. [ABSTRACT FROM AUTHOR]

The study of metal-protein interactions is an expanding field of research investigated by bioinorganic chemists as it has wide applications in biological systems. Very recently, it has been reported that it is possible to study metal-protein interactions by immobilizing biomolecules on metal surfaces and applying experimental approaches based on plasmonics which have usually been used to investigate protein-protein interactions. This is possible because the electronic structure of metals generates plasmons whose properties can be exploited to obtain information from biomolecules that interact not only with other molecules but also with ions in solution. One major challenge of such approaches is to immobilize the protein to be studied on a metal surface with preserved native structure. This review reports and discusses all the works that deal with such an expanding new field of application of plasmonics with specific attention to surface plasmon resonance, highlighting the advantages and drawbacks of such approaches in comparison with other experimental techniques traditionally used to study metal-protein interactions. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

There is a lack of commercially available efficient and autonomous systems capable of continuous monitoring of (bio)chemical data for clinical, environmental, food, or industrial samples. The weakest link in the design of these systems is the (bio)chemical receptor (bCR). The bCR should have transducer ability, the recognition event should be a single reaction, and the bCR should be easily regenerated. Transport proteins and enzymes are well placed as bCR for optical continuous monitoring systems (OCMS). In this paper we review quantitative aspects and the main transducer strategies which have been developed for transport proteins, using periplasmic binding proteins (linking an environmentally sensitive fluorophore or FRET between two fluorophores) and concanavalin A (competitive reversible assays) as representative examples. Efficient immobilization systems and implementation in OCMS are also reviewed. Some kinds of enzymes can fulfil the necessary requirements to be appropriate bCR. Strategies using flavoenzymes chemically modified with fluorophores can be successfully implemented in OCMS and they are, in our opinion, the most appropriate option. [ABSTRACT FROM AUTHOR]

In nonclinical drug development targeting the central nervous system (CNS), the quantitative determination of extracellular brain concentrations of neurotransmitters is a key challenge. In some CNS disorders, the monitoring of the modified profile of neurotransmitter release such as that of histamine may explain the mechanism of action of the drug candidate. Microdialysis is a commonly used method for sampling extracellular levels of neurotransmitters/drug candidates in small laboratory animals. Detection and quantification of extracellular levels of neurotransmitters remain an analytical and technical challenge owing to the low concentrations of neurotransmitters collected, the small microdialysis sample size, and the high amount of inorganic salts. A precolumn derivatization strategy prior to hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry analysis is proposed to quantify histamine release after administration of a CNS research compound. Derivatization using propionic anhydride dissolved in organic solvent combined with the HILIC approach effectively eliminated three time-consuming steps, organic layer transfer, dry down, and reconstitution, all of which are required by traditional reversed-phase liquid chromatography. The formation of propionylated amides, performed under mild conditions, required no further sample cleanup. After a dual microdialysis probe implantation into the prefrontal cortex (neurotransmitters) and in the inferior vena cava of rat (drug candidate), microdialysate fractions were collected every 15 min for 8 h and stored frozen at -20 °C until analysis. The method was validated using 10 μL microdialysate, achieving low limits of quantitation of 83.4 and 84.5 pg.mL for histamine and 1-methylhistamine, respectively. These limits were suitable to assess kinetic release of neurotransmitters and are compatible with those obtained by microdialysis sampling. This method provided the required selectivity, sensitivity, accuracy, and precision to assess release kinetics of histamine and 1-methylhistamine in several hundred rat brain microdialysates after intravenous infusion of CNS drug candidates. [ABSTRACT FROM AUTHOR]

In recent years fluorescence microscopy has become a widely used tool for tissue imaging and spectroscopy. Optical techniques, based on both linear and non-linear excitation, have been broadly applied to imaging and characterization of biological tissues. Among fluorescence techniques used in tissue imaging applications, in recent years two and three-photon excited fluorescence have gained increased importance because of their high-resolution deep tissue imaging capability inside optically turbid samples. The main limitation of steady-state fluorescence imaging techniques consists in providing only morphological information; functional information is not detectable without technical improvements. A spectroscopic approach, based on lifetime measurement of tissue fluorescence, can provide functional information about tissue conditions, including its environment, red-ox state, and pH, and hence physiological characterization of the tissue under investigation. Measurement of the fluorescence lifetime is a very important issue for characterizing a biological tissue. Deviation of this property from a control value can be taken as an indicator of disorder and/or malignancy in diseased tissues. Even if much work on this topic has still to be done, including the interpretation of fluorescence lifetime data, we believe that this methodology will gain increasing importance in the field of biophotonics. In this paper, we review methodologies, potentials and results obtained by using fluorescence lifetime imaging microscopy for the investigation of biological tissues. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Optical coherence tomography (OCT) is a noninvasive imaging technique that provides real-time two- and three-dimensional images of scattering samples with micrometer resolution. By mapping the local reflectivity, OCT visualizes the morphology of the sample. In addition, functional properties such as birefringence, motion, or the distributions of certain substances can be detected with high spatial resolution. Its main field of application is biomedical imaging and diagnostics. In ophthalmology, OCT is accepted as a clinical standard for diagnosing and monitoring the treatment of a number of retinal diseases, and OCT is becoming an important instrument for clinical cardiology. New applications are emerging in various medical fields, such as early-stage cancer detection, surgical guidance, and the early diagnosis of musculoskeletal diseases. OCT has also proven its value as a tool for developmental biology. The number of companies involved in manufacturing OCT systems has increased substantially during the last few years (especially due to its success in opthalmology), and this technology can be expected to continue to spread into various fields of application. [ABSTRACT FROM AUTHOR]

nalysis of the major drugs of abuse in meconium has been established in clinical practice for detecting fetal exposure to illicit drugs, particularly for the ready availability of the sample and ease of collection from diapers, compared with neonatal hair and urine. Very little is known about the occurrence and detection possibilities of therapeutic and licit drugs in meconium. Meconium specimens ( n = 209) were collected in delivery hospitals, from infants of mothers who were suspected to be drug abusers. A targeted analysis method by liquid chromatography–triple quadrupole mass spectrometry (LC-MS/MS) was developed for abused drugs: amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, morphine, codeine, 6-monoacetylmorphine, oxycodone, methadone, tramadol, buprenorphine, and norbuprenorphine. A separate LC-MS/MS method was developed for 11-nor-∆-tetrahydrocannabinol-9-carboxylic acid. A screening method based on LC coupled to time-of-flight MS was applied to a broad spectrum of drugs. As a result, a total of 77 different compounds were found. The main drug findings in meconium were as follows: local anesthetics 82.5% ( n = 172), nicotine or its metabolites 61.5% ( n = 129), opioids 48.5% ( n = 101), stimulants 21.0% ( n = 44), hypnotics and sedatives 19.0% ( n = 40), antidepressants 18.0% ( n = 38), antipsychotics 5.5% ( n = 11), and cannabis 3.0% ( n = 5). By revealing drugs and metabolites beyond the ordinary scope, the present procedure helps the pediatrician in cases where maternal denial is strong but the infant seems to suffer from typical drug-withdrawal symptoms. Intrapartum drug administration cannot be differentiated from gestational drug use by meconium analysis, which affects the interpretation of oxycodone, tramadol, fentanyl, pethidine, and ephedrine findings. [ABSTRACT FROM AUTHOR]

The domestic chicken ( Gallus domesticus) has emerged as a powerful experimental model for studying the onset and progression of spontaneous epithelial ovarian cancer (EOC) with a disease prevalence that can exceed 35% between 2 and 7 years of age. An experimental strategy for biomarker discovery is reported herein that combines the chicken model of EOC, longitudinal plasma sample collection with matched tissues, advanced mass spectrometry-based proteomics, and concepts derived from the index of individuality (Harris, Clin Chem 20: 1535–1542, ). Blood was drawn from 148 age-matched chickens starting at 2.5 years of age every 3 months for 1 year. At the conclusion of the 1 year sample collection period, the 73 birds that remained alive were euthanized, necropsied, and tissues were collected. Pathological assessment of resected tissues from these 73 birds confirmed that five birds (6.8%) developed EOC. A proteomics workflow including in-gel digestion, nanoLC coupled to high-performance mass spectrometry, and label-free (spectral counting) quantification was used to measure the biological intra-individual variability (CV) of the chicken plasma proteome. Longitudinal plasma sample sets from two birds within the 73-bird biorepository were selected for this study; one bird was considered “healthy” and the second bird developed late-stage EOC. A total of 116 proteins from un-depleted plasma were identified with 80 proteins shared among all sample sets. Analytical variability (CV) of the label-free proteomics workflow was measured using a single plasma sample analyzed five times and was found to be ≥CV in both birds for 16 proteins (20%) and in either bird for 25 proteins (31%). Ovomacroglobulin (ovostatin) was found to increase ( p < 0.001) over a 6 month period in the late-stage EOC bird providing an initial candidate protein for further investigation. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Asbestos is a fiber causing lung diseases such as asbestosis and mesothelioma. Although the process involving these diseases remains to be elucidated for developing drugs and treatments, direct consequences of fiber exposure in humans have been clearly demonstrated. These diseases are first characterized by histological heterogeneity and combine chronic inflammation with fibrosis and cellular alterations. As a consequence, asbestosis is usually diagnosed at advanced stages of the disease and treatments are usually inefficient to cure the patients. Here, we review the links established between asbestos fiber chemistry and morphology with the occurrence of associated lung diseases. Cytological and histological aspects of diseases are described with respect to current analytical capabilities, notably for microscopy techniques. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of morbidity and mortality in the United States and cigarette smoking is a primary determinant of the disease. COPD is characterized by chronic airflow limitation as measured by the forced expiratory volume in one second (FEV1). In this study, the plasma proteomes of 38 middle-aged or older adult smokers with mild to moderate COPD, with FEV1 decline characterized as either rapid (RPD, n = 20) or slow or absent (SLW, n = 18), were interrogated using a comprehensive high-throughput proteomic approach, the accurate mass and time (AMT) tag technology. This technology is based upon a putative mass and time tag database (PMT), high-resolution LC separations and high mass accuracy measurements using FT-ICR MS with a 9.4-T magnetic field. The peptide and protein data were analyzed using three statistical approaches to address ambiguities related to the high proportion of missing data inherent to proteomic analysis. The RPD and SLW groups were differentiated by 55 peptides which mapped to 33 unique proteins. Twelve of the proteins have known roles in the complement or coagulation cascade and, despite an inability to adjust for some factors known to affect lung function decline, suggest potential mechanistic biomarkers associated with the rate of lung function decline in COPD. Whether these proteins are the cause or result of accelerated decline will require further research. [ABSTRACT FROM AUTHOR]

An electrospray ionization tandem mass spectrometry (ESI-MS/MS) library which contains over 5,600 spectra of 1,253 compounds relevant in clinical and forensic toxicology has been developed using a hybrid tandem mass spectrometer with a linear ion trap. Pure compound solutions—in some cases solutions made of tablets—were prepared and 1 to 2,000 ng of each compound were injected into the system using standard reversed-phase analytical columns with gradient elution. To obtain maximum mass spectral information enhanced product ion spectra were acquired with positive and/or negative ionization at low, medium, and high collision energies and additionally applying collision energy spread. In this mode, all product ions generated by the different collision energies are trapped in the linear ion trap prior to their detection. The applicability of the library for other types of hybrid tandem mass spectrometers with a linear ion trap of the same manufacturer as well as a standard triple-quadrupole tandem mass spectrometer has been investigated with a selection of compounds. The spectra of the developed library can be used to create methods for target analysis, either screening methods or quantitative procedures by generating transitions for multiple reaction monitoring. For those procedures, suitable transitions and convenient collision energies are selected from the library. It also has been utilized to identify compounds with a multi target screening approach for clinical and forensic toxicology with a standardized and automated system. The novel aspects compared to our former library produced with a standard triple-quadrupole mass spectrometer are the enlargement of the ESI-MS/MS library and the additional acquisition of spectra with collision energy spread. [ABSTRACT FROM AUTHOR]

Sensitive fluorescent probes for the determination of hydrogen peroxide and glucose were developed by immobilizing enzyme horseradish peroxidase (HRP) on Fe3O4/SiO2 magnetic core–shell nanoparticles in the presence of glutaraldehyde. Besides its excellent catalytic activity, the immobilized enzyme could be easily and completely recovered by a magnetic separation, and the recovered HRP-immobilized Fe3O4/SiO2 nanoparticles were able to be used repeatedly as catalysts without deactivation. The HRP-immobilized nanoparticles were able to activate hydrogen peroxide (H2O2), which oxidized non-fluorescent 3-(4-hydroxyphenyl)propionic acid to a fluorescent product with an emission maximum at 409 nm. Under optimized conditions, a linear calibration curve was obtained over the H2O2 concentrations ranging from 5.0 × 10−9 to 1.0 × 10−5 mol L−1, with a detection limit of 2.1 × 10−9 mol L−1. By simultaneously using glucose oxidase and HRP-immobilized Fe3O4/SiO2 nanoparticles, a sensitive and selective analytical method for the glucose detection was established. The fluorescence intensity of the product responded well linearly to glucose concentration in the range from 5.0 × 10−8 to 5.0 × 10−5 mol L−1 with a detection limit of 1.8 × 10−8 mol L−1. The proposed method was successfully applied for the determination of glucose in human serum sample. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Digital polymerase chain reaction (PCR) is a promising technique for estimating target DNA copy number. PCR solution is distributed throughout numerous partitions, and following amplification, target DNA copy number is estimated based on the proportion of partitions containing amplified DNA. Here, we identify approaches for obtaining reliable digital PCR data. Single molecule amplification efficiency was significantly improved following fragmentation of total DNA and bias in copy number estimates reduced by analysis of short intact target DNA fragments. Random and independent distribution of target DNA molecules throughout partitions, which is critical to accurate digital PCR measurement, was demonstrated by spatial distribution analysis. The estimated relative uncertainty for target DNA concentration was under 6% when analyzing five digital panels comprising 765 partitions each, provided the panels contained an average of 212 to 3,365 template molecules. Partition volume was a major component of this uncertainty estimate. These findings can be applied to other digital PCR studies to improve confidence in such measurements. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

In the postgenomic era, information is king and information-rich technologies are critically important drivers in both fundamental biology and medicine. It is now known that single-parameter measurements provide only limited detail and that quantitation of multiple biomolecular signatures can more fully illuminate complex biological function. Label-free technologies have recently attracted significant interest for sensitive and quantitative multiparameter analysis of biological systems. There are several different classes of label-free sensors that are currently being developed both in academia and in industry. In this critical review, we highlight, compare, and contrast some of the more promising approaches. We describe the fundamental principles of these different methods and discuss advantages and disadvantages that might potentially help one in selecting the appropriate technology for a given bioanalytical application. [ABSTRACT FROM AUTHOR]

Point-of-care testing (POCT) is a fast developing area in clinical diagnostics that is considered to be one of the main driving forces for the future in vitro diagnostic market. POCT means decentralized testing at the site of patient care. The most important POCT devices are handheld blood glucose sensors. In some of these sensors, after the application of less than 1 µl whole blood, the results are displayed in less than 10 s. For protein determination, the most commonly used devices are based on lateral flow technology. Although these devices are convenient to use, the results are often only qualitative or semiquantitative. The review will illuminate some of the current methods employed in POCT for proteins and will discuss the outlook for techniques (e.g., electrochemical immunosensors) that could have a great impact on future POCT of proteins. [ABSTRACT FROM AUTHOR]

In terms of testing, modern laboratory medicine can be divided into centralized testing in central laboratories and point-of-care testing (POCT). Centralized laboratory medicine offers high-quality results, as guaranteed by the use of quality management programs and the excellence of the staff. POCT is performed by clinical staff, and so such testing has moved back closer to the patient. POCT has the advantage of shortening the turnaround time, which potentially benefits the patient. However, the clinical laboratory testing expertise of clinical staff is limited. Consequently, when deciding which components of laboratory testing must be conducted in central laboratories and which components as POCT (in relation to quality and timeliness), it will be medical necessity, medical utility, technological capabilities and costs that will have to be ascertained. Provided adequate quality can be guaranteed, POCT is preferable, considering its timeliness, when testing vital parameters. It is also preferred when the central laboratory cannot guarantee the delivery of results of short turn-around-time (STAT) markers within 60 or (even better) 30 min. POCT should not replace centralized medical laboratory testing in general, but it should be used in cases where positive effects on patient care have been clearly demonstrated. [ABSTRACT FROM AUTHOR]

The development of portable instruments for measuring the prothrombin time (PT) in capillary whole blood has made it possible for patients on lifelong coumarin therapy to monitor the effect of the anticoagulant themselves (patient self-testing) and to adjust the dosage if necessary on their own (patient self-management). Dosage variations between individual patients and the narrow therapeutic range of the anticoagulant account for the need to monitor its effect, in order to minimize the risk of thromboembolism resulting from inadequate anticoagulation as well as the risk of bleeding due to overanticoagulation. This review focuses on the prerequisites for the successful implementation of patient self-testing and self-management, such as the selection of eligible patients, appropriate training and the reliability of available instruments. The outcomes of relevant clinical studies involving patient self-testing and self-management are also discussed. [ABSTRACT FROM AUTHOR]

An optimized method based on GC-MS with ethyl chloroformate derivatization has been developed for the comprehensive analysis of endogenous metabolites in serum. Twenty-two reference standards and serum samples were used to validate the proposed method. The correlation coefficient was higher than 0.9900 for each of the standards, and the LOD varied from 125 to 300 pg on-column. The analytical equipment exhibited good repeatability (RSD<10%) for all of the standards. Both the repeatability and the within-48-h stability of the analytical method were satisfactory (RSD<10%) for the 18 metabolites identified in the serum samples. Mean recovery was acceptable for the 18 metabolites, ranging from 70% to 120% with RSDs of less than 10%. Using the optimized protocol and a subsequent multivariate statistical technique, complete differentiation was achieved between the metabolic profile of uremic patients and that of age- and sex-matched normal subjects. Significantly decreased levels of valine, leucine, and isoleucine and increased levels of myristic acid and linoleic acid were observed in the patient group. This work demonstrated that this method is suitable for serum-based metabolic profiling studies. [ABSTRACT FROM AUTHOR]

As researchers attempt to study quorum sensing in relevant clinical or environmental settings, it is apparent that many factors have the potential to affect signaling. These factors span a range of physical, chemical, and biological variables that can impact signal production, stability and distribution. Optimizing experimental systems to natural or clinical environments may be crucial for defining when and where quorum sensing occurs. These points are illustrated in our case study of S. aureus signaling in biofilms, where signal stability may be affected by the host environment. The basic signaling schemes have been worked out at the molecular level for a few of the major quorum-sensing systems. As these studies continue to refine our understanding of these mechanisms, an emerging challenge is to identify if and when the local environment can affect signaling. [ABSTRACT FROM AUTHOR]

Today’s high-resolution mass spectrometers (HRMS) allow bioanalysts to perform untargeted/global determinations that can reveal unexpected compounds or concentrations in a patient’s sample. This could be performed for preliminary diagnosis attempts when usual diagnostic processes and targeted determinations fail. We have evaluated an untargeted diagnostic screening (UDS) procedure. UDS is a metabolome analysis that compares one sample (e.g., a patient) with control samples (a healthy population). Using liquid chromatography (LC)-HRMS full-scan analysis of human serum extracts and unsupervised data treatment, we have compared individual samples that were spiked with one xenobiotic or a higher level of one endogenous compound with control samples. After the use of different filters that drastically reduced the number of metabolites detected, the spiked compound was eventually revealed in each test sample and ranked. The proposed UDS procedure appears feasible and reliable to reveal unexpected xenobiotics (toxicology) or higher concentrations of endogenous metabolites. HRMS-based untargeted approaches could be useful as preliminary diagnostic screening when canonical processes do not reveal disease etiology nor establish a clear diagnosis and could reduce misdiagnosis. On the other hand, the risk of overdiagnosis of this approach should be reduced with mandatory biomedical interpretation of the patient’s UDS results and with confirmatory targeted and quantitative determinations. [ABSTRACT FROM AUTHOR]