Biological Fluid Samples Research Articles

Simultaneous determination of salbutamol and propranolol in biological fluid samples using an electrochemical sensor based on functionalized-graphene, ionic liquid and silver nanoparticles

A simple, rapid, and low-cost electroanalytical method was developed using a glassy carbon electrode modified with functionalized-graphene (FG), ionic liquid (IL) and silver nanoparticles (AgNPs) for simultaneous detection of salbutamol (SAL) and propranolol (PRO). The electrochemical behaviour of the electrodes was investigated by cyclic voltammetry and square wave voltammetry (SWV) under optimised conditions. Using SWV, the AgNP-IL-FG-NF/GCE sensor showed a linear response from 79 nmol L−1 to 2.9 μmol L−1 for SAL and from 0.1 to 2.9 μmol L−1 for PRO with limits of detection of 13 and 17 nmol L−1 for SAL and PRO, respectively. The proposed sensor showed good stability, repeatability and reproducibility, and was applied successfully to the simultaneous determination of SAL and PRO in biological fluid samples. The proposed method proved to be excellent, being therefore a reliable alternative method for the simultaneous determination of SAL and PRO.

Mycophenolate mofetil sensor based on molecularly imprinted polymer/multi-walled carbon nanotubes modified carbon paste electrode

Using square wave voltammetry, a carbon paste electrode modified by molecularly imprinted polymer (MIP) as a recognition element of mycophenolate mofetil (MMF) and multi-walled carbon nanotubes was used for MMF monitoring To investigate the electrode during modification, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were utilized. After optimization of the effective parameters, the anodic peak current of MMF was utilized for dynamic range study which was linear in 9.9 nM–87 μM range. The detection limit of the sensor was 7.0 nM. The capture ability of MIP to target was compared with that of non-imprinted polymer (NIP). The practical application of the sensor in biological fluid samples analysis demonstrates its selectivity, sensitivity, and stability.

The biomimic oxidase activity of layered V2O5 nanozyme for rapid and sensitive nanomolar detection of glutathione

The development of efficient sensing element for rapid and sensitive measurement of glutathione is important on early detection of human diseases. Herein, we introduce the V2O5 nanosheets (NS) as a novel nanozyme to biomimic the oxidase activity toward the rapid detection of glutathione. The 5–6 layered V2O5 NS with 300–600 nm in size is successfully fabricated by ultra-sonication assisted exfoliation of bulk V2O5 using N, N-dimethyl formamide as the intercalation reagent. The sensing mechanism involves the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by V2O5 NS with the concomitant increase in absorption intensity. Upon introduction of glutathione, the oxidized TMB is converted back to its original form and results in the simultaneous decrease in peak intensity at 650 nm. The sensor exhibits a good linear response to glutathione in the range of 10–500 nM with a limit of detection of 2.4 nM. Moreover, the V2O5-TMB sensor exhibits a good selectivity and high specificity, which makes the detection of glutathione applicable in diluted human serum samples. Since no functionalization of the V2O5 NS is required, this sensing system can open a new avenue to serve as an efficiently biomimic oxidase for glutathione detection in biological fluid samples.

Information technology and hardware-software complex for monitoring the state of biological fluid samples

The article deals with the development of information technology for monitoring and diagnostics of the state of liquid biological samples. The method is proposed on the basis of factor analysis caused by an information electric signal as a result of mechanical action on a biological sample. The paper provides a detailed analysis of the methods using a similar (piezoelectric) effect in medical and research practice. The aim of the work was the development and experimental study of the method and systems for monitoring the state of liquid biological samples. The basic models are used as the informational model of the factorial influence i.e. parametric and the model of dispersion components. The level of mechanical action on the liquid sample is used as the levels of the influencing factor in this method, and the potential difference from the electrodes immersed in the liquid sample was used as an informative signal. The paper shows the structural diagram of a laboratory installation and describes the principle of its operation. For experimental verification of the developed method, 10 samples of whole blood of patients (5 without pathology, 5 with colorectal cancer) were used in the work. The robot provides a technique for preparing the data obtained and provides an information technology for identifying informative features. With the use of the described informative models of factorial influence, an informative analysis of the proposed parameters and an estimation of the quantitative content of control information in them are presented. As a result of the experimental study, it was concluded that the developed information technology makes it possible to detect changes in the state of the controlled biochemical material with 95 % certainty.

Extracellular Vesicles in Oncology: Progress and Pitfalls in the Methods of Isolation and Analysis.

The possibility to study solid tumors through the analysis of extracellular vesicles in biological fluids is one of the most exciting and rapidly advancing field in cancer research. The extracellular vesicles are tiny sacs released in both physiological and pathological conditions and can be used to monitor the evolution of several pathological states, including neoplastic diseases. Indeed, these vesicles carry biological informations and can affect the behavior of recipient cells by transferring proteins, DNA, RNA, and microRNA. In this review the authors analyze the methods to collect biological fluid samples (urine, plasma/serum, and cell supernatant), and to isolate and quantify extracellular vesicles highlighting advantages and drawbacks. Moreover, the authors provide an overview on the adoption and the advantages of the methods (such as digital PCR, next generation sequencing, reverse-phase protein microarrays, flow-cytometry, etc.) most frequently used to analyze the molecular content of extracellular vesicles. Despite the great scientific interest on this topic, there is still a great uncertainty about which is the best method for the collection, isolation, quantification, and molecular evaluation of these vesicles and a standardization is needed. The features of EVs make them ideal candidates for liquid biopsy-based biomarkers. However, the small size of EVs makes their analysis very difficult and requires multiple advanced technologies, being therefore a limitation.

Dielectrophoretic Nanoparticle Aggregation for On-Demand Surface Enhanced Raman Spectroscopy Analysis.

Rapid chemical identification of drugs of abuse in biological fluids such as saliva is of growing interest in healthcare and law enforcement. Accordingly, a label-free detection platform that accepts biological fluid samples is of great practical value. We report a microfluidics-based dielectrophoresis-induced surface enhanced Raman spectroscopy (SERS) device, which is capable of detecting physiologically relevant concentrations of methamphetamine in saliva in under 2 min. In this device, iodide-modified silver nanoparticles are trapped and released on-demand using electrodes integrated in a microfluidic channel. Principal component analysis (PCA) is used to reliably distinguish methamphetamine-positive samples from the negative control samples. Passivation of the electrodes and flow channels minimizes microchannel fouling by nanoparticles, which allows the device to be cleared and reused multiple times.

Circulating Micrornas Predict Survival of Patients with Tumors of Glial Origin

The World Health Organization has recently introduced molecular prognostic-diagnostic biomarkers in the classification of Central Nervous System (CNS) tumors. In order to characterize subclasses of tumors that cannot find a precise location in the current classification, and, or cannot be tested because of scant material, it is important to find new molecular biomarkers in tissue and, or biological fluid samples. In this study, we identified serum microRNAs that could serve as biomarkers for the diagnosis and prognosis of patients with tumors of glial origin. We retrospectively analyzed microRNA expression in the serum extracellular vesicles of patients with tumors of glial origin. Extracellular vesicles RNA was analyzed by Nanostring. qRT-PCR confirmed 6 overexpressed microRNAs: hsa-miR-4443, hsa-miR-422a, hsa-miR-494-3p, hsa-miR-502-5p, hsa-miR-520f-3p, and hsa-miR-549a. Hsa-miR-4443 was the only microRNA that showed significant differences in most comparisons. In situ hybridization (ISH), confirmed that our signature was mostly expressed in cancer cells.Importantly, hsa-miR-549a and hsa-miR-502-5p expression predicted prognosis in patients with tumors of glial origin. Although more studies are needed, we demonstrated that serum vesicles microRNA profiles are promising diagnostic and prognostic molecular biomarkers that will find an actual application in the clinical practice of CNS tumors.

Design of hierarchical electrocatalytic mediator for one step, selective screening of biomolecules in biological fluid samples

Active electrocatalytic electrode designs are needed for the sensitive and selective detection of a single or multi-active biomolecule among biological components. We report the design of hierarchical NiO catalyst (HNC) for one-step monitoring of bioactive molecules such as ascorbic acid (AA), dopamine (DA), and uric acid (UA). The novel mesostructured geometries, active surface sites, and multi-diffused spaces for easy electron movement through gaps provide highly active electrocatalytic electrode designing surface. Controlled HNC architecture along electrode-design surface domains having double-head branches spread out along both sides of the dipole-like rod may lead to the vital electron transfer and fast response signaling of multi-bioactive molecules in one-shot triggering individually or simultaneously. Electrochemical analyses showed evidence that the proposed electrode design can detect each component up to 0.02 µM. Sensitive detection up to 1.127, 0.02, and 0.978 µM and wide-range responses of 25–800, 2–60, and 10–000 µM for AA, DA, and UA, respectively, were observed. The simultaneous monitoring and selective signaling of AA, DA, and UA in real urine samples in one step under different potentials were realized. Thus, the HNC-modified electrode can monitor and evaluate the coexistence of biomolecules simultaneously in multi-components. The electrochemical oxidation of AA, DA, and UA at the HNC-modified electrode and the corresponding DPV peaks with losing of 2e−/2H+.

An efficient sample preparation method based on dispersive liquid–liquid microextraction associated with back extraction for trace determination of acidic pharmaceuticals

Reduction of matrix effect seems to be a great challenge for the development of a practical method in bioanalysis. In this regard, a simple and efficient DLLME procedure along with a back-extraction step (DLLME-BE) was developed for the preconcentration of four common non-steroidal anti-inflammatory drugs (NSAIDs) in various biological fluid samples. Briefly, the analytes of interest were initially transferred into the extraction solvent followed by the back-extraction into an immiscible basic methanol (as an acceptor phase) for further preconcentration and clean-up. The main purpose of the work is reducing the matrix effect and sensitive determination of target molecules in the complex matrices. Following on, the separation and determination of the analytes were carried out using GC–MS (in-port derivatization) and HPLC-DAD instrument. The influential parameters affecting the DLLME-BE method were evaluated in detail and the best extraction conditions were established. Under the optimum conditions, low method detection limits in the range of 0.1–1.0 and 0.1–6.0 µg L−1 were obtained for GC–MS and HPLC-DAD analysis, respectively. Additionally, fair intra-day precisions of 2.7–14.5 and 2.8–7.8% as well as inter-day precisions of 3.9–14.5 and 3.5–8.1% were achieved for the GC–MS and HPLC-DAD analysis, respectively. Finally, the method was successfully applied for the determination of four common NSAIDs in different biological fluid samples.

One-pot synthesis of Au-Cu2O/rGO nanocomposite based electrochemical sensor for selective and simultaneous detection of dopamine and uric acid

A Au-Cu2O/rGO nanocomposite based electrochemical sensor for selective and simultaneous detection of dopamine and uric acid was developed. The nanocomposite was prepared by a one-pot synthesis method and the resulted product was characterized by transmission electron microscope (TEM), energy dispersive X-ray (EDX) analysis, X-ray diffraction (XRD) patterns and X-ray photoelectron spectroscopy (XPS). From TEM image, the presence of Au-Cu2O supported on rGO sheets were identified. The XRD analysis reveals the face centered cubic (FCC) structure of Au-Cu2O nanoparticles and the XPS spectrum showed the presence of the constituent elements in the nanocomposite. The electrochemical studies on the Au-Cu2O/rGO nanocomposite towards dopamine (DA) and Uric Acid (UA) were tested using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The CV studies showed enhanced catalytic activity for dopamine due to the synergistic effect of the ternary nanocomposite which was also further supported by the increased surface roughness of the material. From DPV, the detection limit of 3.9 μM and 6.5 μM and linear range of 10–90 μM and 100–900 μM was estimated for DA and UA. The selective and simultaneous detection of DA and UA was demonstrated. The sensor shows long term stability and reproducibility. Quantification of DA and UA in biological fluid samples (ie human serum and urine) has also been carried out to prove the practical applicability of the sensor.

The multiplex analysis of drug medicinals on the basis of technology of immunochips Phosphan.

The new technique of multiplex qualitative analysis of narcotic, psychotropic remedies is developed on the basis of technology Phosphan using immunochips in the format of standard 96-wells plates, monoclonal antibodies to narcotic compounds and Pt-coproporphyrin as a long luminescent marker. The multiplex analysis was implemented using 20 mkl of human biological fluid (urine, blood serum or saliva) of 2 discs of 3.2 mm in diameter made of dried urine spot on paper. No preliminary processing or dilution of analyzed sample is required. The large range of measured concentrations was demonstrated under high sensitivity of analysis: 1 ng/ml of morphine and methadone, 0.5 ng/ml of barbiturates, 2 ng/ml of benzoylecgonine, methamphetamine, cannabinoids and benzodiazepines, 8 ng/ml amphetamine at variability of results no more than 15%. The approbation of technique was implemented using valid samples of urine (n=197) and blood serum (n=98) demonstrated that the technique permits to detect properly opiates, cocaine, cannabinoids, methadone, benzodiazepine, barbiturates and amphetamines at absence of false positive results in case of analysis of samples containing non-narcotic medications. The results of study of samples of dried urine spot on paper (n=50) well coincided with the results of analysis of fluid samples for all analyzed analytes. On the basis of proposed multiplex analysis a test-system Narc-Phosphan was developed for quantitative studying simultaneously up to 96 samples of various biological fluids, including as dried spots on paper. The analysis demonstrated high sensitivity, specificity and exactness during detection of the most prevailed narcotic substances that permits to propose this technique as a primary test during mass check-ups of population with purpose of detection of drug abuse, especially at the earlier stage.

Uptake of Tyrosine Amino Acid on Nano-Graphene Oxide.

Graphene oxide (GO) is emerging as a promising nanomaterial with potential application in the detection and analysis of amino acids, DNA, enzymes, and proteins in biological fluid samples. So, the reaction of GO with amino acids should be characterized and determined before using it in biosensing methods and devices. In this study, the reaction of tyrosine amino acid (Tyr) with GO was characterized using FT-IR, UV-vis spectrophotometry, and scanning electron microscopy (SEM) before its use. The optimum conditions for GO’s interaction with Tyr amino acid have been studied under variable conditions. The optimum conditions of pH, temperature, shaking time, and GO and tyrosine concentrations for the uptaking of tyrosine amino acid onto the GO’s surface from aqueous solution were determined. The SEM analysis showed that the GO supplied was in a particle size range between 5.4 and 8.1 nm. A pH of 8.4–9.4 at 25 °C and 5 min of shaking time were the optimum conditions for a maximum uptake of 1.4 μg/mL of tyrosine amino acid onto 0.2 mg/mL of GO.

A multi-walled carbon nanotube-based magnetic molecularly imprinted polymer as a highly selective sorbent for ultrasonic-assisted dispersive solid-phase microextraction of sotalol in biological fluids.

A modified multiwalled carbon nanotube-based magnetic molecularly imprinted polymer (MWCNT-MMIP) was synthesized and applied for selective extraction and preconcentration of sotalol (SOT) in biological fluid samples by using ultrasonic-assisted dispersive solid-phase microextraction (UA-DSPME). The synthetic particles were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Brunauer-Emmett-Teller (BET) analysis, vibrating sample magnetometry (VSM) and Fourier transform infrared spectroscopy (FTIR). The screening of UA-DSPME was preliminarily performed by Plackett-Burman design (PBD) and, subsequently, central composite design (CCD) under response surface methodology (RSM) was used individually for evaluation of the significant factors and their possible interaction effects on the adsorption process. Batch mode adsorption studies were performed to evaluate the adsorption kinetics, adsorption isotherms, and selective recognition of MWCNT-MMIPs. The adsorption equilibrium of SOT using MWCNT-MMIPs could be well-defined with the Langmuir isotherm model and the maximum adsorption capacity was calculated to be 79.36 mg g-1. Under optimized conditions, the SOT was selectively and effectively extracted in real biological samples and good linearity was obtained with correlation coefficients (R2) over 0.996 and the detection limit (S/N = 3) was 0.31 ng mL-1. The average recoveries of the spiked human urine and plasma samples at four concentration levels of SOT ranged from 94.60-102.50 and 97.40-101.60 percent, respectively, and the relative standard deviation was found to be lower than 4.50%. The results illustrated that the proposed MWCNT-MMIPs@UA-DSPME extraction method coupled with HPLC-UV determination could be applied for sensitive and selective analysis of trace SOT in biological fluid samples.

Detection and scavenging of hydroxyl radical via D-phenylalanine hydroxylation in human fluids

Hydroxyl radical (.OH) is highly reactive, and therefore very short-lived. Finding new means to accurately detect .OH, and testing the ability of known .OH scavengers to neutralize them in human biological fluids would leverage our ability to more effectively counter oxidative (.OH) stress-mediated damage in human diseases. To achieve this, we pursued the evaluation of secondary products resulting from .OH attack, using a detection system based on Fenton reaction-mediated D-phenylalanine (D-Phe) hydroxylation. This reaction in turn generates o-tyrosine (o-tyr), m-tyrosine (m-tyr) and p-tyrosine (p-tyr). Here, these isomers were separated by HPLC, equipped with fluorescence detectors due to the natural fluorescence of these hydrotyrosines. By extension, we found that, adding radical scavengers competed with D-Phe on .OH attack, thus allowing to determine the .OH quenching capacity of a given compound expressed as inhibition ratio percent (IR%). Using a kinetic approach, we then tested the .OH scavenging capacity (OHSC) of well-known antioxidant molecules. In a test tube, N,N′-dimethylthiourea (DMTU) was the most efficient scavenger as compared to Trolox and N-Acethyl-L-cysteine, with NAC being the less effective. OHSC assay was then applied to biological fluid samples as seminal plasma, human serum from normal subjects and patients undergoing hemodialysis (HD), colostrum and human breast milk from mothers that received daily doses of 30g of chocolate (70% cocoa) during pregnancy. We found that a daily administration of dark chocolate during pregnancy almost doubled OHSC levels in breast milk (1.88 ± 0.12 times, p < 0.01). Furthermore, HD treatment determined a significant reduction of serum OHSC concentration (54.63 ± 2.82%, p < 0.001). Our results provide evidence that Fenton reaction-mediated D-Phe hydroxylation is a suitable method for routine and non-invasive evaluation of .OH detection and its scavenging in human biological fluids.

Evaluation of semi-automatic image analysis tools for cerebrospinal fluid electrophoresis of IgG oligoclonal bands

BackgroundIgG concentrations in cerebrospinal fluid generally range from 20 to 45 mg/L. In multiple sclerosis immune reactions lead to intrathecal synthesis of specific IgGs that can be detected in biological fluid samples both quantitatively and qualitatively by isoelectric focusing of supplementary oligoclonal IgG bands. MethodA simple tool, using the MATLAB application, to facilitate and improve isoelectric focusing profile analysis is presented and evaluated in terms of its sensitivity, repeatability and reproducibility. A comparison between human readers and semi-automatic method has also been performed. ResultsResults from the semi-automatic method were found to be equivalent or superior to generally employed laboratory methods. Repeatability analysis for semi-automatic processing yielded coefficients of variation (CVs) in the 3–7% range, and using a sample with an estimated IgG concentration of 200 mg/L, four bands were still visible after dilution to 5 mg/L, corresponding to band concentrations of 1.1–1.6 mg/L. Discordances between visual inspection and automatic analysis only appear at threshold levels for interpretation (the gray zone). ConclusionThe semi-automatic method has acceptable performance for routine implementation.

Point-spread function engineering enhances digital Fourier microscopy.

While numerous optical methods exist to probe the dynamics of biological or complex fluid samples, in recent years digital Fourier microscopy techniques such as differential dynamic microscopy have emerged as ways to efficiently combine elements of imaging and scattering methods. Here, we demonstrate, through experiments and simulations, how point-spread function engineering can be used to extend the reach of differential dynamic microscopy.

FORENSIC MEDICAL EVALUATION OF TOXICOLOGICAL EFFECTS OF ETHANOL AMONG CHILDREN

This article contains brief literature review of Russian and foreign literature dedicated to problem of toxicological effects of ethanol among children, statistical data and autopsy results of children with ethanol in blood samples, results of forensic chemical and chemical-toxicological investigation of biological fluids samples from children and teenagers from 0 to 17 in Bureau of Forensic Medicine of Moscow Region during the period from 2003 to 2017.

Tissue distribution of ochratoxin A in pigs after administration of two-levels contaminated diets

The aim of the present study was to determine the levels of ochratoxin A (OTA) in pigs experimentally exposed to this mycotoxin and to evaluate if bile may be used to assess exposure to OTA. Twelve hybrid pigs were divided into 3 equal groups, a control group D0, and 2 experimental groups, D1 fed with 50 µg OTA/kg diet, and D2 fed with 500 µg OTA/kg diet for 15 days. At the end of the test, the animals were euthanized and samples of different tissues and biological fluids were analysed by HPLC-fluorescence detection for the presence of OTA. Samples of unconventional edible tissues such as lung and heart were also taken for analysis because they are used in typical Italian regional dishes. The Italian guidance value for OTA of 1 µg/kg established for pork meat and derived products has been exceeded in all the matrices from both the experimental groups. The comparison between OTA levels detected in D1 and D2 groups showed clearly a linear dose-response relationship. Based on the mean values measured, OTA distribution follows the order blood plasma &gt; lung &gt; kidney (in D1 group), heart (in D2 group) &gt; heart (in D1 group), kidney (in D2 group) &gt; bile &gt; liver &gt; fat &gt; muscle. Analysis of bile can be useful for the detection of OTA in pigs. However, since blood can easily be taken from pigs, and given the correlation between the mycotoxin concentration detected in this matrix and the concentrations detected in the others, OTA level in blood is a more viable approach to assessing the presence of OTA in edible tissues. As lung and heart may contain high concentrations of OTA, the analytical controls should also include these matrices.

Highly sensitive and selective detection of mercury ions using N, S-codoped graphene quantum dots and its paper strip based sensing application in wastewater

The development of robust and low-cost sensing materials with superior selectivity and sensitivity for rapid detection of analytes in a wide variety of samples has attracted much attention. Herein, the N, S-codoped graphene quantum dots (N, S-GQDs) with high quantum yield were fabricated by one-pot hydrothermal method for highly sensitive and selective detection of nanomolar level of mercury ions (Hg2+) in water and wastewater. The as-prepared N, S-GQDs are uniform in size with mean particle size of 3.5±0.5nm. The doping of nitrogen atoms increases the quantum yield to 41.9%, while the introduction of sulfur atoms enhances the selectivity of Hg2+ via strong coordination interaction. The fluorescence intensity of N, S-GQDs is quenched proportionally after adding Hg2+ and a dynamic range of 4 orders of magnitude with limit of detection of 0.14nM is obtained in deionized water. The N, S-GQDs nanosensing probes can be successfully applied to the sewage and dye wastewater samples and a linear range of 0.1–15μM with recovery of 96–116% is obtained. In addition, the coating of N, S-GQDs onto paper strip provides an excellently rapid screening and highly selective technique for Hg2+ detection in real wastewater. Results obtained in this study clearly indicate that the N, S-GQDs are promising nanomaterials which can open an new avenue to design the GQD based sensing probe for highly sensitive and selective detection of metal ions and other analytes in environmental and biological fluid samples.

The Analysis of Phenylbutazone and Its Active Metabolite, Oxyphenbutazone, in Equine Tissues (Muscle, Kidney, and Liver), Urine, and Serum by LC-MS/MS.

This study reports the use of two validated LC with tandem MS (MS/MS) methods to study the residue depletion profile of phenylbutazone (PBZ) and its metabolite oxyphenbutazone (OXPBZ) from equine serum, urine, and muscle, kidney, and liver tissues. One LC-MS/MS method, with an LOQ of 1.0 ng/mL for PBZ and 2.0 ng/mL for OXPBZ, was used for the analysis of the two drugs in the biological fluids (equine urine and serum); the other LC-MS/MS method, with an LOQ of 0.5 ng/g for PBZ and OXPBZ, was used for the analysis of the drugs in the equine tissue samples. PBZ was administered intravenously to two horses dosed with 8.8 mg/kg PBZ once daily for 4 days and sacrificed humanely at a slaughter plant 7 days after the last drug administration. Urine, serum, and kidney, liver, and muscle tissues were collected from the two horses and shipped on ice to the laboratory and stored at -20°C until analysis. The concentrations of PBZ and OXPBZ residues in the biological fluid and tissue samples collected at slaughter were measured with the two validated LC-MS/MS methods using deuterated internal standards. The results demonstrate that the validated methods are fit for studying the depletion kinetics of PBZ residues in equine tissues and biological fluids.