Biological Fluid Samples Research Articles

The sensitive fluorescence assay of phosphates and alkaline phosphatase based on terbium nanocomplexes synthesized via ligand proportion regulation

Bimetallic dual-ligands terbium nanocomplexes (Tb/DPA/Fe/SSA NCs) with unique internal structure was fabricated by in situ coordination assembly of the Tb3+, Fe3+ and antenna ligands of 2,6-pyridine dicarboxylic acid (DPA) and sulfosalicylic acid (SSA). Interestingly, the regulation of internal ligand SSA proportion endows the nanocomplex with unique structural heterogeneity, which leads to excellent sensing specificity. The formed terbium nanocomplex 1 (NC1) with low SSA ligand (nSSA:nFe3+=1) contributed to the detection of phosphate (PO43-), and its high degree of freedom competes for the internal metal sites of the loose structure NC1, thus recovering the fluorescence of NC1. The enhanced fluorescence intensity of NC1 shows a wide linear relationship with the concentration of PO43- in the range of 1–280 μmol/L and 280–640 μmol/L, and the detection limit is 0.3 μmol/L. Terbium nanocomplex 2 (NC2), where high proportion of SSA ligand (nSSA:nFe3+=6) promotes NC2 to be a coordination saturated nanocomplex, has an extraordinary fluorescence response to pyrophosphate(PPi) instead of PO43-, and PPi with multidentate coordination structure can compete for metal sites in NC2, resulting in enhanced fluorescence. Utilizing the difference of fluorescence response to PPi and PO43-, NC2 serves as a fluorescent probe to detect ALP and exhibits detection limit of 0.26 U/L. As fluorescence probe, Tb/DPA/Fe/SSA NCs have the outstanding merits in simple preparation and rapid response, which has been accomplished for PO43- and ALP analysis in biological fluid samples.

A new nanocomposite-based screen-printed graphene electrode for sensitive and selective detection of 8-hydroxy-2′-deoxyguanosine

8-Hydroxy-2′-deoxyguanosine (8-OHdG) is a crucial marker used to appraise the degree of endogenous oxidative DNA damage in the human body. For routine clinical analysis, a simple, rapid, cost-effective, reliable, selective, sensitive, and portable approach is exceedingly required. In this work, we first reported on the fabrication of an electrochemical sensor based on poly(L-methionine) and gold nanoparticle-modified screen-printed graphene electrode (poly(L-Met)/AuNPs/SPGE) for the sensitive and selective detection of 8-OHdG in the presence of uric acid at a normal level in urine, which is the major interference when using square wave voltammetry. Sodium phosphate buffer solution containing sodium chloride and sodium dodecyl sulfate was used as the supporting electrolyte for specific detection of 8-OHdG. The oxidation peaks of 8-OHdG and uric acid can be obviously separated. Under optimal conditions, the linearity between the anodic peak current and the 8-OHdG concentration was obtained within the range of 1–50 µM, and the limit of detection (3SD/Slope) and limit of quantification (10SD/Slope) were found to be 92 and 306 nM, respectively. This fabricated sensor was successfully applied to determine the 8-OHdG concentration in biological fluid samples with good selectivity, sensitivity, reliability, reproducibility, accuracy, and precision. The promising results demonstrated that this proposed methodology could be useful for medical personnel in diagnosing the disease risk from 8-OHdG levels at an early stage.

Synthesis of a Novel [diresorcinate‐1,10‐phenanthrolinecobalt(II)] Complex, and Potentiodynamic Fabrication of Poly(DHRPCo)/GCE for Selective Square Wave Voltammetric Determination of Procaine Penicillin G in Pharmaceutical and Biological Fluid Samples

AbstractThis work presents synthesis, and characterization of a novel [diresorcinate‐1,10‐phenanthrolinecobalt(II)] (DHRPCo) complex, and potentiodynamic fabrication of poly(DHRPCo)/GCE for SWV determination of procaine penicillin G (ProPenG) in various pharmaceutical and biological samples. While UV‐Vis and FT‐IR results demonstrated stepwise synthesis of DHRPCo, the CV and EIS results evidenced modification of the surface of GCE by an electroactive polymer film that improved electrode surface area. In contrast to the peak of ProPenG at the unmodified electrode, an irreversible oxidative peak at poly(DHRPCo)/GCE with lower potential and six folds of current enhancement observed, that confirmed electrocatalytic effect of the polymer, SWV response of poly(DHRPCo)/GCE showed linear dependence on concentration of ProPenG in the range 0.1–200 μM, with LoD of 4.9 nM. Spike recovery results in various pharmaceutical and biological samples in the range 98.03–106.50 %, interference recovery in the presence of selected potential interferents at their various levels with associated errors under 3.39 %, and stability of the modifier with analysis time validated the method for determination of ProPenG. Poly(DHRPCo)/GCE was applied for determination of ProPenG in three brands of injection pharmaceutical formulation, human urine, human blood serum, and cow's milk samples. While no ProPenG was detected in the human urine, serum, and cow's milk samples, detected ProPenG in the injection powder samples was within 96.1–101.3 % of company label. The developed method showed superior performance over recently reported methods making it an excellent candidate for determination of ProPenG in real samples.

A Highly Sensitive Modified Glassy Carbon Electrode with a Carboxylated Multi-walled Carbon Nanotubes/Nafion Nano Composite for Voltammetric Sensing of Dianabol in Biological Fluid.

The extraordinary prerequisite for the analysis of an anabolic steroid, namely dianabol (DB), has inspired towards the development of a cost-effective and high-performance sensing probe. Thus, a simple and robust electrochemical sensor (c-MWCNTs-Nafion®lGCE) for dianabol (DB), a widely used steroid, was developed using a glassy carbon electrode (GCE) modified with functionalized carboxylated multi-walled carbon nanotubes (c-MWCNT) and Nafion®. At pH 7 - 8, differential pulse-cathodic stripping voltammetry (DP-CSV) displayed two cathodic peaks at -0.85 and -1.35 V that varied linearly over a wide range (9.0 × 10-9 (2.7 μg L-1) - 9.0 × 10-6 (2.7 × 103 μg L-1) mol L-1) and 2.9 × 10-6 (8.7 × 102 μg L-1) - 8.0 × 10-5 (2.4 × 104 μg L-1) mol L-1) of DB concentrations, respectively. The low limits of detection and quantification at peak I (-0.85 V) were 2.7 × 10-9 (8.1 × 10-1 ng mL-1) and 9.0 × 10-9 (2.7 ng mL-1) mol L-1, respectively. The repeatability and reproducibility displayed relative standard deviations lower than 5%. The method was applied for DB analysis in human urine and subsequently compared with the standard HPLC method. Interference of common metabolites in biological fluids samples to DB sensing was insignificant. This method has distinctive advantages e.g. precise, short analytical time, sensitive, economical, reproducible and miniaturized sample preparation for DB analysis in biological samples of human origin.

Backbone extension via peptidomimetics at N-terminal; self-assembled nanofibrous cluster and application to selective progesterone detection in an aqueous medium

Despite the adequacy of the endogenous steroid (progesterone) levels in biological functioning, elevated levels of progesterone hormone have several physiological effects that are amplified due to its direct and indirect uptake from the environment, food products, and medical therapy. So, it is much needed to evaluate the progesterone levels in environmental samples as well as for biological fluids. In this work, we focused on the development of the nano sensing probe for the selective detection of progesterone among the library of steroid hormones belonging to the class of female sex hormones. Herein, functionalization of dipeptide is carried out at N-terminal to produce N-functionalized dipeptide (SS3), and simultaneously, its self-assembly properties are explored. Furthermore, HR-TEM imaging was also performed to examine the morphology of the self-assembled architectures before and after the addition of the steroid hormone. To investigate the binding mechanism of the sensing probe, Fluorescence spectroscopy, Circular Dichroism (CD), MD-Simulation, and DFT studies were performed and studied in detail. Moreover, to check the potency of the real-time application of the developed nanoprobe, we have successfully determined the spiked concentration of progesterone levels in pharmaceutical and biological fluid samples with functional percentage recovery. Also, the stability and other competitive binding studies of the probe with the coexisting substances are performed to check the rationality of the sensing probe at physiological conditions.

Amperometric biosensors for L-arginine and creatinine assay based on recombinant deiminases and ammonium-sensitive Cu/Zn(Hg)S nanoparticles

There are limited data on amperometric biosensors (ABSs) based on deiminases that produce ammonium as a byproduct of enzymatic reaction. The most frequently proposed biosensors utilizing such a mode are based on potentiometric transducers, which contain at least two enzymes in the bioselective layer; this complicates the procedure and increases the cost of analysis. Thus, the construction of a one-enzyme ABS is a practical problem. In our manuscript ABSs for the direct measurement of creatinine (Crn) and l-arginine (Arg), based on the recombinant bacterial creatinine deiminase (CDI) and arginine deiminase (ADI), are described. To choose the best chemosensor on ammonium ions, a number of nanoparticles (NPs) were synthesized and characterized using cyclic voltammetry. Hybrid Cu/Zn(Hg)S-NPs, having a good selectivity and an extremely high sensitivities towards ammonium ions (5660 A M−1 m−2 at +170 mV and 1870 A M−1 m−2 at −300 mV, respectively), was selected for the development of deiminase-based ABSs. The novel biosensors exhibited very high sensitivities (2660 A M−1 m−2 to Crn for CDI-ABS; 1570 A M−1 m−2 to Arg for ADI-ABS), broad linear ranges, low limits of detection, satisfactory storage stabilities and good selectivities towards natural substrates. The constructed CDI-ABS and ADI-ABS were tested on real samples of biological fluids and juices for Crn and Arg assay, respectively. High correlations of the obtained results with the reference methods were demonstrated for the target analytes.

Electrochemical sensors for the detection of SARS-CoV-2 virus

Coronavirus (COVID-19), a deadly pandemic has spread worldwide and created many global health issues. Though methods of its detection are being continuously developed for the early detection and monitoring of COVID-19, still there is need for more novel methods. The presently used methods include rapid antigen tests, serological surveys, reverse transcription-polymerase chain reaction (RT-PCR), artificial intelligence-based techniques, and assays based on sensors/biosensors. Of all these, RT-PCR test has high sensitivity and specificity though it requires more time for testing and need for skilled technicians. Recently, electrochemical sensors have been developed for rapid monitoring and detection of SARS-CoV-2 from the patient’s biological fluid samples. This review covers the recently developed electrochemical sensors that are focused on the detection of viral nucleic acid, immunoglobulin, antigen, and the entire viral particles. In addition, we also compare and assess their detection limits, sensitivities and specificities for the identification and monitoring of COVID-19. Furthermore, this review will address the best practices for the development of electrochemical sensors such as electrode fouling, limit of detection/limit of quantification determination and verification.

BSA-stabilized manganese phosphate nanoflower with enhanced nanozyme activity for highly sensitive and rapid detection of glutathione

The development of an efficient protein-inorganic nanohybrid with superior nanozyme activity for highly sensitive detection of glutathione (GSH) is essential for early diagnosis of human diseases. Herein, a rapid and highly sensitive colorimetric assay using self-assembled bovine serum albumin-hydrated manganese phosphate nanoflowers (MnPNF) as a biomimic oxidase is developed for GSH detection in human serum. The BSA can complex with Mn2+ to serve the nucleation center to produce MnPNF in the presence of phosphate-buffered saline (PBS). The morphology and surface characterization results show that the MnPNF is assembled with hierarchical nanoplates to form 500 nm nanoflowers. The oxidase-like activity of MnPNF is based on the redox reaction with 3,3′,5,5′-tetramethylbenzidine. However, the addition of GSH can reduce MnPNF to Mn2+, and subsequently supresses the oxidase-like activity and a yellow color at 450 nm is observed in the presence of H2SO4. The MnPNF-based nanozyme exhibits excellent sensing ability toward GSH detection, and a good linear relationship between the change in absorbance at 450 nm and the added amounts of GSH at 50 nM–10 μM with low limits of detection of 20 and 26.6 nM in the PBS and diluted human serum, respectively, is observed. Moreover, the sensing probe shows a superior selectivity over the other 16 interferences, which drive the determination of GSH feasible in real human serum. Since the MnPNF can be simply prepared at room temperature and no functionalization is required, this assay can be used to design the highly efficient biomimic oxidase for effective sensing of GSH and other disease-related biomolecules in biological fluid samples.

Use of methods of the biocrystallomics in personification of the treatment with high diluted drugs

Background: One of most important trends in modern medicine is its transformation to personalized diagnostics and treatment. This tendency fully applies to homeopathy, especially considering that this discipline is based on the principles of individual approach to the patient. At the same time, the methods of individualization of treatment in homeopathy are relatively few. 
 Aims: Develop of methodology and methods of application biocrystalloscopic tests to personalize the homeopathic treatment. The proposed technology became previously proposed for ozone therapy practice as biocrystallomics pre-test. It includes study of the result of co-crystallization of a biological liquid of a patient with the intended dose of the drug. At the same time, the character of co-crystallization of biological liquid with several doses of tested drug or different parameters of the action of the studied factor is comparatively estimated for the purpose of individualization. Most appropriate for a particular patient believe the dose that causes optimal structuring in the dried microscopic slides obtained from a mixture of biological fluid and a solution containing a given dose of the drug. The comparison is made with a control sample of biological fluid, which has not been exposed to any effects. The optimal result is a sample that matches the control sample as much as possible. It is most preferably for biocrystallomics pre-test to use blood serum or plasma as an analyzed biological fluid.
 Methodology: To study the characteristics of the structuring of the semicroscopic slides we use the previously created system of semi-quantitative parameters. They are calculated on a straight four-point scale and include: crystallizability (semi-quantitative indicator of crystallization activity), structure index (complexity criterion for constructing crystal elements of facias), facia destruction degree (indicator of “correctness” of crystal formation) and clearity of facia marginal zone (parameter indicating the amount of native protein in the sample of biological fluid). These indicators comprehensively characterize all the main features of the process of dehydration structurization of biological substrate.
 Conclusion: It should be noted that the technique described above is universal and can be applied to any liquid or soluble compounds with potential therapeutic activity, including their ultra-high dilutions. The data obtained by us indicate the possibility of using this technology in homeopathy.

Thread-based isotachophoresis coupled with desorption electrospray ionization mass spectrometry for clean-up, preconcentration, and determination of alkaloids in biological fluids

A thread-based isotachophoresis method coupled with desorption electrospray ionization mass spectrometry (TB-ITP-DESI-MS) was developed and applied for clean-up, preconcentration, and determination of alkaloids in biological fluids. This simple approach enables the focusing and rapid analysis of analytes of interest in complex matrices that are otherwise challenging using direct ambient mass spectrometry. The TB-ITP platform components were rapidly and reproducibly fabricated at low-cost using 3D printing. A single string of nylon 6 thread was used as the electrophoresis substrate and a cotton knot, tied to the nylon thread, was used as the trapping zone of the ITP focused model analytes (coptisine, berberine and palmatine). The trapping efficiency was evaluated upon different commercially available threads with different chemical properties and cotton was selected as the best material due to its highest trapping efficiency and subsequent DESI-MS ionization efficiency. Up to 11.6-fold increase in signal to noise ratio (S/N) was obtained using the proposed method compared to direct DESI-MS detection, due to the reduced matrix interference and focusing. The results demonstrated that the TB-ITP-DESI-MS approach is a viable solution for the analysis of complicated biological fluid samples.

Finger-Actuated Microneedle Array for Sampling Body Fluids

The application of microneedles (MNs) for minimally invasive biological fluid sampling is rapidly emerging, offering a user-friendly approach with decreased insertion pain and less harm to the tissues compared to conventional needles. Here, a finger-powered microneedle array (MNA) integrated with a microfluidic chip was conceptualized to extract body fluid samples. Actuated by finger pressure, the microfluidic device enables an efficient approach for the user to collect their own body fluids in a simple and fast manner without the requirement for a healthcare worker. The processes for extracting human blood and interstitial fluid (ISF) from the body and the flow across the device, estimating the amount of the extracted fluid, were simulated. The design in this work can be utilized for the minimally invasive personalized medical equipment offering a simple usage procedure.

Determination of Folic Acid Using Biosensors-A Short Review of Recent Progress.

Folic acid (FA) is the synthetic surrogate of the essential B vitamin folate, alternatively named folacin, pteroylglutamic acid or vitamin B9. FA is an electroactive compound that helps our body to create and keep our cells healthy: it acts as the main character in a variety of synthetic biological reactions such as the synthesis of purines, pyrimidine (thus being indirectly implied in DNA synthesis), fixing and methylation of DNA. Therefore, physiological folate deficiency may be responsible for severe degenerative conditions, including neural tube defects in developing embryos and megaloblastic anaemia at any age. Moreover, being a water-soluble molecule, it is constantly lost and has to be reintegrated daily; for this reason, FA supplements and food fortification are, nowadays, extremely diffused and well-established practices. Consequently, accurate, reliable and precise analytical techniques are needed to exactly determine FA concentration in various media. Thus, the aim of this review is to report on research papers of the past 5 years (2016–2020) dealing with rapid and low-cost electrochemical determination of FA in food or biological fluid samples.

Developed rapid spectrophotometric method for simultaneous quantitative determination of metformin and linagliptin mixture as antidiabetic drugs by artificial intelligence methodology in biological fluid and pharmaceutical sample

In this study, simultaneous measurement of metformin (MET) and linagliptin (LIN) present in synthetic mixtures, antidiabetic tablet, and the biological sample was studied by chemometrics methods along with spectrophotometric technique. These procedures involved feedforward-backpropagation neural network (FFBP-NN) with Levenberg–Marquardt (LM) and comprehensive Grobner bases (CGB) algorithms, as well as least square support vector machine (LS-SVM). The mean square error (MSE) of the LM algorithm was obtained 1.22 × 10−28 (layer=2 with 10 neurons) and 1.93 × 10−27 (layer=3 with 8 neurons) for MET and LIN, respectively. In the CGB algorithm, layer 2 with 10 neurons for both components was introduced as the best layer and neurons with the least error. The optimal parameters of the LS-SVM method, including regularization (γ) and width (σ) parameters with the root mean square error (RMSE) of 0.4416 and 0.1634 were found for MET and LIN, respectively. The proposed methods were implemented on antidiabetic tablet and the results were compared with high-performance liquid chromatography (HPLC) using analysis of variance (ANOVA) test. The urine sample was also studied as a complex matrix by these methods. A combination of spectrophotometry and chemometrics can be used to improve the quality of pharmaceutical products due to its fast and low-cost and no need for the separation step.

Use of reduced graphene oxide for sensitive determination of sulfanilamide in synthetic biological fluids and environmental samples by batch injection analysis

In this work, a reduced graphene oxide sensor associated to the batch injection analysis system with amperometric detection was investigated for the simple, fast and sensitive monitoring of sulfanilamide in lake water and synthetic biological fluid (saliva, sweat and urine) samples. The modified sensor showed a potential displacement of 170 mV and an increase in peak current of 5.5-fold for the electrochemical oxidation of sulfanilamide in comparison to the bare electrode, resulting in a highly sensitive and selective method. Besides, the developed method showed excellent analytical performance with a limit of detection of 2.30 µmol L−1, linear range from 10 to 50 µmol L−1, relative standard deviation below 5.6%, and recovery ranges varying from 83 to 109% for the analysis of spiked samples. In addition, the minimum sample preparation (simple dilution) combined with a high analytical frequency (130 analyses per hour) can be highlighted. Thus, the proposed method demonstrates to be a promising analytical tool for the implementation in routine analysis of clinical interest and environmental monitoring in loco.

Validation of the biochemical method for determining the total cholesterol level in the samples of biological fluids as a base for providing the quality of dyslipidemia diagnostics

The main principles of creating a quality system in modern laboratory diagnostics are: standardization of laboratory processes by developing standard operating procedures; general quality management of laboratory research based on the development and implementation of the requirements of international standards (according to ISO 15189: 2015 "Medical laboratories. Basic requirements for quality and competence"); quality control of all stages of the laboratory process through the implementation of the validation procedure.
 Aim. To develop a methodology for conducting validation procedures to assess the suitability of a biochemical method for determining the level of total cholesterol in biological fluids in the Clinical Diagnostic Laboratory of the Clinical Diagnostic Center of the NUPh.
 Materials and methods. The object of the study was a standardized method for determining the concentration of total cholesterol. The method was validated using the “Cholesterol Reagent Set” test kit and the standard sample “Chemical control. Reagent kit. Level 1 ", manufactured by High Technology, Inc. (USA) with known concentration. The measurements were carried out on an Express Plus automatic biochemical analyzer manufactured by Bayer Corporation, Germany. When processing the research results, descriptive statistics were used and a number of statistical evaluations were carried out.
 Results. A protocol and a validation report were developed at the Clinical Diagnostic Laboratory of the CDC NUPh to assess the suitability of the method for determining the concentration of cholesterol in biological fluids by the photometric enzymatic method on an automatic biochemical analyzer Express Plus (using reagents and control material manufactured by High Technology, Inc., USA). The validation characteristics of the method were determined: repeatability and reproducibility, correctness and uncertainty of measurements.
 Evaluation of the internal laboratory repeatability and reproducibility of this technique indicates the absence of gross errors in the operation of the instrument and statistically important differences in measurements. The assessment of the correctness of the method (carried out using the control material) proved that the systematic error is not significant (according to a given acceptance criterion). The expanded uncertainty calculation showed that the obtained values ​​of the total cholesterol level can be considered accurate and reliable.
 Conclusions. Validation of the method for determining total cholesterol in human blood by the photometric enzymatic method has proven that this method has performance characteristics that correspond to the regulated ones, meets the established criteria, and the parameters measured with it correspond to the proper ones.
 Key words: validation, determination method, total cholesterol, repeatability and reproducibility, correctness and uncertainty of measurements

Synthesis of homogeneously distributed gold nanoparticles built-in metal free organic framework: Electrochemical detection of riboflavin in pharmaceutical and human fluids samples

This study describes the synthesis of gold nanoparticles (AuNPs) incorporated melamine based covalent organic framework (MCOF) and their modification on glassy carbon electrode (GCE) for the sensitive and selective detection of riboflavin (RF). The MCOF was synthesized by refluxing melamine and terephthalaldehyde followed by AuNPs were incorporated into MCOF. The synthesized nanocomposite (MCOF-AuNPs) was confirmed by different spectral, microscopic and electrochemical methods. The TEM images show spherical AuNPs with an average diameter of 8 nm on the sheet-like structure of MCOF. The heterogeneous electron transfer rate constant (ket) was calculated for 1 mM K3/K4[Fe(CN)6] containing 0.1 M KCl at bare GC, MCOF and MCOF-AuNPs/GCEs was found to be 1.14, 1.72 and 4.31 × 10-4 cm2 s−1, respectively. The higher ket value achieved at MCOF-AuNPs/GCE indicates that the electron transfer rate reaction was faster at this electrode than others. The MCOF-AuNPs modified electrode was then utilized for the reduction of RF in 0.2 M phosphate-buffered saline (PBS) (pH 7.0). The MCOF-AuNPs/GCE showed 3.0 and 1.7-fold higher reduction current with 40 and 60 mV less positive potential shift against RF than bare GCE and MCOF/GCE. Under the optimized sensing parameters, the linear range concentration was achieved from 5 nM to 0.2 mM with the LOD of 2.0 nM. Finally, the present sensor was applied for the quantitative determination of RF in human biological fluids samples (human blood, urine, and saliva). Besides, the practical application was also demonstrated by determining RF in vitamin B-complex tablets and satisfactory results were obtained.

Semen impairment and occurrence of SARS-CoV-2 virus in semen after recovery from COVID-19.

STUDY QUESTIONHow is the semen quality of sexually active men following recovery from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection?SUMMARY ANSWERTwenty-five percent of the men with recent SARS-Cov-2 infections and proven healing were oligo-crypto-azoospermic, despite the absence of virus RNA in semen.WHAT IS KNOWN ALREADYThe presence of SARS-CoV-2 in human semen and its role in virus contagion and semen quality after recovery from coronavirus disease 2019 (COVID-19) is still unclear. So far, studies evaluating semen quality and the occurrence of SARS-CoV-2 in semen of infected or proven recovered men are scarce and included a limited number of participants.STUDY DESIGN, SIZE, DURATIONA prospective cross-sectional study on 43 sexually active men who were known to have recovered from SARS-CoV2 was performed. Four biological fluid samples, namely saliva, pre-ejaculation urine, semen, and post-ejaculation urine, were tested for the SARS-CoV-2 genome. Female partners were retested if any specimen was found to be SARS-CoV-2 positive. Routine semen analysis and quantification of semen leukocytes and interleukin-8 (IL-8) levels were performed.PARTICIPANTS/MATERIALS, SETTING, METHODSQuestionnaires including International Index of Erectile Function and Male Sexual Health Questionnaire Short Form were administered to all subjects. The occurrence of virus RNA was evaluated in all the biological fluids collected by RT-PCR. Semen parameters were evaluated according to the World Health Organization manual edition V. Semen IL-8 levels were evaluated by a two-step ELISA method.MAIN RESULTS AND THE ROLE OF CHANCEAfter recovery from COVID-19, 25% of the men studied were oligo-crypto-azoospermic. Of the 11 men with semen impairment, 8 were azoospermic and 3 were oligospermic. A total of 33 patients (76.7%) showed pathological levels of IL-8 in semen. Oligo-crypto-azoospermia was significantly related to COVID-19 severity (P < 0.001). Three patients (7%) tested positive for at least one sample (one saliva; one pre-ejaculation urine; one semen and one post-ejaculation urine), so the next day new nasopharyngeal swabs were collected. The results from these three patients and their partners were all negative for SARS-CoV-2.LIMITATIONS, REASONS FOR CAUTIONAlthough crypto-azoospermia was found in a high percentage of men who had recovered from COVID-19, clearly exceeding the percentage found in the general population, the previous semen quality of these men was unknown nor is it known whether a recovery of testicular function was occurring. The low number of enrolled patients may limit the statistical power of study.WIDER IMPLICATIONS OF THE FINDINGSSARS-CoV-2 can be detected in saliva, urine, and semen in a small percentage of men who recovered from COVID-19. One-quarter of men who recovered from COVID-19 demonstrated oligo-crypto-azoospermia indicating that an assessment of semen quality should be recommended for men of reproductive age who are affected by COVID-19.STUDY FUNDING/COMPETING INTEREST(S)None.TRIAL REGISTRATION NUMBERN/A.

Phenylalanine-responsive fluorescent biosensor based on graphene oxide-chitosan nanocomposites catalytic film for non-destructive fish freshness grading

An optical enzymatic phenylalanine biosensor for non-invasive evaluation of fish freshness using biofluid from the fish body surface has been developed based on phenylalanine dehydrogenase (PDH) and nicotinamide adenine dinucleotide (NAD+) chemisorbed on the graphene oxide-chitosan (GO/CT) nanocomposites film. The immobilized PDH on the biosensor surface via EDC/NHS coupling strategy catalyzed the oxidative deamination of phenylalanine to phenylpyruvate, and concomitantly converted the cofactor NAD+ (oxidized form) to fluorophore NADH (reduced form), which can be quantified by using fluorescence spectrophotometer. The phenylalanine-responsive fluorescent biosensor showed a dynamic linear concentration range between 0.03 mM and 0.24 mM phenylalanine at 644 nm emission wavelength with an excitation wavelength of 320 nm. The limit of detection (LOD) of the fluorometric phenylalanine biosensor was estimated at 0.02 mM with a fast response time of 1 min. No significant interference by amines-synthesized amino acids, such as histamine, creatine, sarcosine, creatinine, tyrosine and uric acid at typical physiological levels commonly co-exist with phenylalanine in the biofluid metabolite of fish towards fresh fish quality grading. Validation study of the GO/CT catalytic film revealed excellent practicality of the biosensor in biological fluid sample of tuna fish (Euthynnus affinis) for detection of phenylalanine. The proposed GO/CT nanocomposites biofilm offers rapid test that can be assessed with an easy manner to rapidly analyze large number of samples in a short period of time.

The Synthesis of Molecularly Imprinted Polymers on Microcentrifuge Tube Filters for Solid-Phase Extraction and the HPLC-UV Determination of Andrographolides.

Molecular imprinting-based solid-phase extraction has been in the spotlight to improve recognition selectivity and detection sensitivity of andrographolides. The synthesis of molecularly imprinted polymers on micro centrifuge tube filters for the extraction and the determination of andrographolides were investigated. Molecularly imprinted polymers were synthesized using the photo-polymerization method for the preconcentration of andrographolides (AD) template molecule using 2,2-dimethoxy-2-phenylacetophenone as initiators, the mixture of 1-dodecanol and toluene solvent, 2-hydroxyethyl methacrylate and ethylene glycol dimethacrylate as functional monomers and cross-linked, respectively. The resultant AD molecularly imprinted polymers (AD-MIPs) were characterized using the Fourier-transform infrared spectrum and scanning electron microscopy. The maximum adsorption of AD-MIPs toward the andrographolides was found to be 85%, and could reach binding equilibrium within 60min. The sample solution was separated by AD-MIP using solid-phase extraction (SPE). Subsequently, the sample solution was analyzed by the high-performance liquid chromatography (HPLC) method. The AD-MIP could be successfully applied to specifically separate and determine the andrographolides from pharmaceutical products and biological fluid samples with relatively high recoveries (102.01-108.61%). The present method is simple, selective, accurate, and provides a promising alternative to traditional SPE sorbents for the extraction and determination of andrographolides in real samples and biological fluid samples.

DEVELOPMENT OF A SAMPLE PREPARATION PROCEDURE OF BIOLOGICAL FLUIDS BY SOLID-PHASE EXTRACTION OF PYRROLIDINOPHENONE DERIVATIVES

The topical issue of forensic toxicology is the development of sample preparation procedures that provide a high level of selectivity and evidence of the results by gas chromatography-mass spectrometric analysis (GC-MS) with a quadrupole detector. The aim of the study is to develop a technique for sample preparation of biological fluids by solid-phase extraction (SPE) using a model compound phenylpiracetam and to measure its metrological characteristics. Materials and methods. The objects of the study were model samples of biological fluids (blood and urine) with a phenylpiracetam concentration of 20.0-6000.0 ng / ml. The SPE procedure was developed by modifying the separation technique on mixed-type sorbents used in routine practice for screening studies for drugs, including pyrrolidinophenone derivatives. Isopropyl alcohol was used as an eluent for desorption of phenylpiracetam from the molecularly imprinted sorbent. Results. Comparison of the average recovery of phenylpiracetam (for concentration range of 20.0-6000.0 ng / ml) from aqueous solutions, blood and urine samples shows that the recovery from model blood samples is 6% lower than the recovery from aqueous solutions and 4% lower compared to urine samples. The relative errors in determining the degree of extraction from blood samples used to assess the correctness of the sample preparation procedure did not exceed ± 12% for all levels of 20 Евразийский Союз Ученых (ЕСУ) #12(81), 2020 &#x0D; phenylpiracetam concentrations. For the upper concentration level (1500.0 ng / ml), the error is ± 11.5%. The minimum value was observed at an average concentration level of 500.0 ng / ml and amounted to ± 5.3%. The relative errors in determining the degree of extraction from urine samples for all concentration levels did not exceed ± 6%. For the upper concentration level (1500.0 ng / ml), the error is about ± 4%. The precision of the results of phenylpiracetam determining recovery from model blood samples at the lower level of concentrations was 16.1%, at the upper level of concentrations - 6.1%. For urine samples, the precision of the recovery at the lower concentration level was 12.6%, at the upper concentration level - about 5%.