Simulated Urine Samples Research Articles

Luminescent carbon dots endowed with selective recognition of the carcinoid tumor biomarkers in biological fluids

Biomarkers are commonly used in both basic research and clinical practice as diagnostic tools. Particularly, cancer biomarkers can be applied to the early detection and diagnosis or screening the tumors. Of special note, malignant tumors can release serotonin (5-hydroxytryptamine, 5-HT). In fact, 5-HT is an excellent biomarker for carcinoid tumors since its level in blood plasma is hardly affected by other parameters. Moreover, secretion of 5-HT gives rise to elevated levels of 5-hydroxyindole-3-acetic acid (HIAA), the major metabolite of 5-HT, in urine samples of the patients with carcinoid tumors. 5-HT and HIAA are regarded as eligible biomarkers, of which concentrations in blood plasma and urine samples can be utilized for prognosis, and early diagnosis of carcinoid syndrome. Here, we unveil the facile synthesis and characterization of new Tb(III)-doped carbon dots (CDs) in the present work. Strikingly, CDs exhibited superior features for sensing 5-HT and HIAA. It is of worth to note that the Tb(III)-doped CDs induce phosphorescent response to both 5-HT and HIAA in water. Limit of detections (LODs) for 5-HT and HIAA were found to be 0.44 nM and 0.49 nM, respectively. Remarkably, these are the lowest reported values for luminescent systems to date. Moreover, CDs were utilized as chemosensors for 5-HT and HIAA in simulated blood plasma and synthetic urine samples, respectively. To this purpose, extensive luminescence measurements were performed. Accordingly, the obtained results attested that CDs were promising chemosensors for the detection of both 5-HT and HIAA in simulated blood plasma and synthetic urine samples, respectively. Last but not least, we successfully demonstrate that Tb(III)-doped CDs can be used for the detection of 5-HT in artificial cerebrospinal fluid as well as in conditions mimicking the environment within secretory vesicles where 5-HT is stored. To the best of our knowledge, this is the first example of CDs which can induce remarkable phosphorescent response to 5-HT and HIAA in biological fluids.

Electrochemical Sensor Based on Electrodeposited Zinc-Aluminium Layered Double Hydroxide Modified Glassy Carbon Electrode for Chlorpromazine Sensing

Chlorpromazine functions as a potent dopamine D2 receptor antagonist, leading to adverse motor-related effects encompassing cataracts, musculoskeletal disorders, alterations in eyelid pigmentation, muscle contractions, and tremors-addressing the need for a reliable analytical tool, an electrodeposited thin film was synthesized on a glassy carbon electrode (GC) surface. This film comprised zinc-aluminium layered double hydroxide (Zn-Al LDH), with nitrate ions intercalated between the LDH layers. The electrocatalytic behavior of the resulting electrode (GC/Zn-Al LDH) in the oxidation and reduction of nitrogen and sulfur atoms within the thiazine ring structure of chlorpromazine was systematically studied using cyclic voltammetry. Evaluation of the electrode’s analytical response through diverse electroanalytical techniques demonstrated that the square wave voltammetry-assisted electrochemical sensor displayed a broad detection range for chlorpromazine (1 × 10−4 to 1 mM), with a sensitivity of 91.86 μA mM−1 and an impressive low detection limit of 16 × 10−6 mM. Furthermore, the performance of the developed electrode was assessed in detecting and quantifying chlorpromazine levels in simulated human urine samples through recovery studies. The results indicated satisfactory recovery rates, affirming the efficacy of the Zn-Al LDH-modified GC electrode. Noteworthy features of the electrochemical sensor included high surface coverage, improved electron transfer rate, reliable repeatability, and exceptional reproducibility. These characteristics collectively contribute to the sensor’s popularity for accurately detecting and quantifying of chlorpromazine in real-world samples.

Highly sensitive and selective dopamine determination using a photoelectrochemical sensor based on the BiVO4 and surface molecularly imprinted poly(acrylic acid-co-TRIM) on vinyl functionalized carbon nanotubes

This paper describes the development of a new photoelectrochemical (PEC) sensor for dopamine (DA) determination based on the nanocomposite constituted of a molecularly imprinted poly(acrylic acid-co-TRIM) functionalized onto MWCNT with vinyl groups (MWCNT-MIP) physically mixed with monoclinic bismuth vanadate. Synthesized polymeric materials were characterized by FT-IR, TGA, SEM and TEM. The sensor based on glassy carbon electrode modified with BiVO4/MWCNT-MIP provided a limit of detection (LOD) of 31.1 nmol/L and a linear range from 103.6 to 2500 nmol/L. The proposed sensor exhibited selectivity to DA in competitive analysis with epinephrine, norepinephrine, ascorbic acid, citric acid, uric acid, creatine and urea, and it was applied for analysis of DA in simulated urine samples using external calibration curve with accuracy attested by recovery values between 91 and 106 %.

Development and pre-clinical evaluation of a Zika virus diagnostic for low resource settings.

Zika virus (ZIKV) is a re-emerging flavivirus that poses a significant public health threat. ZIKV exhibits a wide array of non-vector borne human transmission routes, such as sexual transmission, transplacental transmission and blood transfusion. Detection and surveillance of ZIKV is considered paramount in prevention of major outbreaks. With the majority of cases reported in low-resource locations, simple, low-cost detection methods are considered highly desirable. Here we have developed a sensitive and specific ZIKV diagnostic using reverse transcription recombinase-aided amplification (RT-RAA) coupled with lateral flow detection (LFD) targeting a highly conserved region of the ZIKV NS1 gene. We show our rapid, isothermal-ZIKV-diagnostic (Iso-ZIKV-Dx) can detect 500 copies of synthetic ZIKV RNA/μL in under 30 min at a constant 39°C. Using simulated urine samples, we observed that Iso-ZIKV-Dx also detects as low as 34.28 RNA copies/reaction of ZIKV (MR766 strain). Specificity testing confirmed that our test does not detect any co-circulating flaviviruses (dengue, West Nile, Japanese encephalitis, Murray Valley encephalitis and yellow fever viruses) or chikungunya virus. Sample processing results show complete inactivation of ZIKV (MR766 strain) in 5 min at room temperature using our novel viral RNA sample preparation reagent. Furthermore, lateral flow strips testing demonstrates positive diagnoses in as little as 5 min in running buffer. Contrary to conventional RT-qPCR, our Iso-ZIKV-Dx does not require expensive machinery, specialised laboratory settings or extensively trained personnel. Pre-clinical evaluation demonstrates that our test exhibits robust, in-field capabilities without compromising sensitivity or specificity. When compared to the gold-standard RT-qPCR, our Iso-ZIKV-Dx test offers an array of applications that extend beyond diagnostics alone, including potential for surveillance and monitoring of ZIKV vector competency.

Establishment and application of a point-of-care testing and diagnosis method for early immediate expression gene IE1 of cytomegalovirus in maternal urine based on isothermal amplification

BackgroundHuman Cytomegalovirus virus (HCMV) is a worldwide virus that causes no serious symptoms in most adults. However, HCMV infection during pregnancy, it may lead to a series of serious complications, such as hearing loss, mental retardation, visual impairment, microcephaly and developmental retardation. AimThe aim of this study was to develop a simple, low dependence on equipment and accurate method for HCMV detection based on the recombinase polymerase amplification (RPA) and lateral flow chromatography strip (LFS) reading. MethodsIn order to meet the feasibility of HCMV early screening, three pairs of RPA primers were designed based on the UL123 gene encoding IE1, which was expressed immediately in the early stage of HCMV. In order to improve the specificity of the reaction and satisfy the visual detection, a specific probe was designed to insert THF site between upstream and downstream primers, fluorescein isothiocyanate (FITC) and C3spacer were used to modify the 5′ end and the 3′ end respectively, and Biotin was used to modify the 5′ end of the reverse primer. HCMV standard strain AD169 was enriched by ARPE-19 cells culture, and its genome was extracted. The primers and probes were screened by RPA-LFS test, and the optimal reaction temperature and time were determined The specificity was verified in different viruses, bacteria and parasites. The standard curve was drawn based on the constructed recombinant plasmid of pMD18T-HCMV-UL123 and used for HCMV genomic DNA quantification and determination of the detection sensitivity. Urine samples from artificial HCMV contamination or clinical collection were prepared to evaluate the consistency with the results of real-time quantitative PCR. ResultsThe results showed that the primers and probes for HCMV RPA-LFS detection based on UL123 gene were successfully screened, the amplification of HCMV genomic DNA with as low as 30 copies could be completed at 37 °C within 15 min, it did not react with Human herpesvirus 1, Streptococcus pyogenes, Candida albicans, Listeria monocytogenes, Y. enterocolitica, Klebsiella Pneumoniae, Enterobacter cloacae, Citrobacter freundii, Vibrio alginnolyfificus, Vibrio parahaemolyticus, S. typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa and Trichomonas vaginalis. The positive rate of PCR was 96.67 % in 30 simulated urine samples and 100 % in 127 clinical urine samples with the same UL123 gene detection. ConclusionsTo sum up, we developed a diagnostic method for HCMV based on UL123 gene combined with RPA and LFS, which is low dependent on equipment, fast, sensitive and specific, provide reference for point-of-care testing HCMV in grass-roots laboratories and remote areas.

One-step Nonenzymatic Electrochemical Sensor for the Detection of Sarcosine Using Nanozyme Glutathione Copper Complex

Background: The present study aims to develop the use of glutathione copper complex for the detection of sarcosine, a marker for prostate cancer. The glutathione-copper complex was successfully synthesized at room temperature and characterized using FTIR, UV, and Scanning Electron Micrograph. Methods: The structure of the glutathione copper complex was found to be a 1:2 Metal: Ligand ratio. The cyclic voltammogram for the glutathione-copper complex modified electrode showed an oxidation peak around -0.037 V, which indicates the irreversible oxidation of copper ions. The addition of sarcosine decreased the oxidation potential of the glutathione copper complex indicating the ability of the working electrode, for the detection of sarcosine in the sample using differential pulse voltammetry. Results: The results indicated that the glutathione-copper complex modified electrode revealed good sensitivity, selectivity, and linearity against the detection of sarcosine in the range from 0.1 μM to 2.5 μM. The LOD and LOQ were calculated using a linear prediction model, the data obtained from differential pulse voltammetry technique for known and simulated urine samples was 0.72 μM, 5.13 μM, and 1.45 μM, 39.94 μM, respectively. The sensitivity for the developed working electrode obtained for the known and simulated sample was 0.0567 μA/μM and 0.02913 μA/μM, respectively. Conclusion: Thus, we concluded that the glutathione copper complex decorated glassy carbon electrode is a good candidate for the detection of sarcosine with good selectivity and sensitivity for real-time monitoring.

Pillar[n]MaxQ sensing ensemble: Application to world anti-doping agency banned substances

The molecular recognition properties of two pillar[n]arene sulfates (P[5]AS and P[6]AS) toward a panel of world anti-doping agency banned substances (1–11) were investigated by a combination of isothermal titration calorimetry, 1H NMR spectroscopy, and molecular modelling. Subsequently a sensing ensemble based on indicator displacement was created using the P[5]AS•lucigenin and P[6]AS•Hoechst 33258 complexes which allowed differentiation among the analytes with 90% accuracy. The assay was extended to allow the quantitation of pseudoephedrine in simulated urine samples with a limit of quantitation that is 30-fold below the WADA threshold.

Intelligent Colorimetric Sensor for Discriminative Detection of Pd2+/Pd0 and PPh3 at Different Wavelength Readouts

AbstractIn this report, we presented our results on a recognition of multiple analytes using trisubstituted PDI‐based chemosensor DNP in 50 % HEPES buffered‐CH3CN solution. Upon addition of Pd2+ or Pd0, DNP showed near IR ratiometric changes (A759nm/A560nm) with detection limit of 283 nM and 190 nM, respectively. These spectroscopic changes are associated with color change from violet to light yellow (de‐propargylation). The de‐propargylation reaction of DNP is faster in the presence of Pd2+ than Pd0. Upon addition of PPh3, DNP resulted in appearance of new bands at 615 and 698 nm with decrease of absorbance intensity at 560 nm with detection limit of 349 nM. Two isosbestic points at 504 nm and 600 nm was observed. This change is associated with color changes from violet to blue. DNP coated TLC‐strips can be used for the detection of 4×10−4 M Pd0 and PPh3. We have demonstrated the practical application of DNP (i) for the detection of Pd0 in live A549 cells and (ii) for detection of Pd2+/Pd0 in simulated urine (10 %) samples with recovery of (>98±5 %).

A Study of the Detection of SARS-CoV-2 by the Use of Electrochemiluminescent Biosensor Based on Asymmetric Polymerase Chain Reaction Amplification Strategy.

A new and reliable method has been constructed for detecting severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) open reading frames 1ab (ORF1ab) gene via highly sensitive electrochemiluminescence (ECL) biosensor technology based on highly efficient asymmetric polymerase chain reaction (asymmetric PCR) amplification strategy. This method uses magnetic particles coupled with biotin-labeled one complementary nucleic acid sequence of the SARS-CoV-2 ORF1ab gene as the magnetic capture probes, and [Formula: see text]-labeled amino-modified another complementary nucleic acid sequence as the luminescent probes, and then a detection model of magnetic capture probes-asymmetric PCR amplification nucleic acid products-[Formula: see text]-labeled luminescent probes is formed, which combines the advantages of highly efficient asymmetric PCR amplification strategy and highly sensitive ECL biosensor technology, enhancing the method sensitivity of detecting the SARS-CoV-2 ORF1ab gene. The method enables the rapid and sensitive detection of the ORF1ab gene and has a linear range of 1-[Formula: see text] copies/[Formula: see text], a regression equation of [Formula: see text] = [Formula: see text] + 2919.301 ([Formula: see text] = 0.9983, [Formula: see text] = 7), and a limit of detection (LOD) of 1 copy/[Formula: see text]. In summary, it can meet the analytical requirements for simulated saliva and urine samples and has the benefits of easy operation, reasonable reproducibility, high sensitivity, and anti-interference abilities, which can provide a reference for developing efficient field detection methods for SARS-CoV-2.

CRISPR-assisted test for Schistosoma haematobium

Schistosomiasis is a major neglected tropical disease targeted for elimination as a public health issue by 2030, however there is an urgent need for more sensitive and specific diagnostic tests suitable to resource-limited settings. Here we developed CATSH, a CRISPR-assisted diagnostic test for Schistosoma haematobium, utilising recombinase polymerase amplification, Cas12a-targeted cleavage and portable real-time fluorescence detection. CATSH showed high analytical sensitivity, consistent detection of a single parasitic egg and specificity for urogenital Schistosoma species. Thanks to a novel CRISPR-compatible sample preparation developed using simulated urine samples containing parasitic eggs, CATSH had a sample-to-result within 2 h. The components of CATSH can be lyophilised, reducing cold chain dependence and widening access to lower and middle-income countries. This work presents a new application of CRISPR diagnostics for highly sensitive and specific detection of parasitic pathogens in remote areas and could have a significant impact on the elimination of neglected tropical diseases.

Two Co-Based Metal–Organic Framework Isomers with Similar Metal-Carboxylate Sheets: Turn-On Ratiometric Luminescence Sensing Activities toward Biomarker N-Acetylneuraminic Acid and Discrimination of Ga3+ and In3+

Two supramolecular Co-MOF isomers, namely, {[Co(L)0.5(m-bimb)]·3H2O}n (LCU-115) and {[Co(L)0.5(p-bimb)]·3H2O}n (LCU-116), were synthesized from an amide-containing carboxylic acid N,N″-(3,5-dicarboxylphenyl)benzene-1,4-dicarboxamide (H4L) and two flexible positional isostructural N-containing ligands m-bimb and p-bimb (m-bimb = 1,3-bis((1H-imidazol-1-yl)methyl)benzene; p-bimb = 1,4-bis((1H-imidazol-1-yl)methyl)benzene). The carboxylate ligands connect Co(II) centers to form 2D metal-carboxylate sheets, which are extended further by m-bimb and p-bimb to form a 2D bilayer with parallel stacking (LCU-115) and a 3D framework (LCU-116), respectively. Luminescence measurements indicated that these two complexes exhibited interesting multiresponsive sensing activities toward pH, biomarker N-acetylneuraminic acid, and trivalent cations Ga3+/In3+. They show highly sensitive turn-on fluorescence responses in the acidic range and can also be regarded as on-off-on vapoluminescent sensors to typical acidic and basic gases HCl and Et3N. It is worth noting that these complexes have excellent turn-on ratiometric fluorescence sensing ability for N-acetylneuraminic acid (NANA) with detection limits as low as 7.39 and 8.06 μM, respectively. Furthermore, they were successfully applied for the detection of NANA in simulated urine and serum samples with satisfactory results. For ion detection, LCU-116 could detect both Ga3+ and In3+, while LCU-115 could distinguish Ga3+ from In3+ with the latter showing luminescence quenching. The sensing mechanism was investigated in detail by XRD, UV-vis, EDS, XPS, SEM, and TEM. The results of interday and intraday precision studies gave low RSD values in the range of 1.19-3.53%, ascertaining the reproducibility of these sensors. The recoveries for the sensing analytes in simulated urine/serum or real water are satisfactory from 96.7 to 103.3% (toward NANA) and 96.6 to 115.0% (toward Ga3+ and In3+), indicating that these two complexes also possess acceptable reliability for monitoring in real samples. The results indicated that the supramolecular isomers LCU-115 and LCU-116 are promising material candidates for application in biological and environmental monitoring.

Use of newly synthetized magnetic Fe3O4 nanoparticles modified with hexadecyl trimethyl ammonium bromide for the sensitive analysis of antidepressant drugs, duloxetine and vilazodone in wastewater and urine samples.

A new enrichment and determination method involving HPLC-DAD analysis following magnetic solid-phase extraction (MSPE) was developed to detect trace amounts of two antidepressant drugs, namely, duloxetine (DUL) and vilazodone (VIL). In this study, a solid-phase sorbent was newly synthesized for use in the MSPE and its characterization was carried out by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy and X-ray diffraction (XRD) techniques. In this proposed method, DUL and VIL molecules were enriched using newly synthesized magnetic-based nanoparticles in the presence of pH 10.0 buffer and desorbed with acetonitrile to a smaller volume prior to chromatographic determinations. After experimental variables were optimized, the VIL and DUL molecules were analyzed at wavelengths of 228 nm for DUL and 238 nm for VIL with isocratic elution of methanol, trifluoroacetic acid (TFA) (0.1%), and acetonitrile (10 : 60 : 30). The detection limits obtained under optimized conditions were 1.48 ng mL-1 and 1.43 ng mL-1, respectively. The %RSD values were found to be lower than 3.50% with model solutions containing 100 ng mL-1 (N:5). Finally, the developed method was successfully applied to wastewater samples and simulated urine samples, and quantitative results were obtained in the recovery experiments.

Determination of Melatonin Using a New Integrated Electrochemical Platform of Functionalized Multi-Walled Carbon Nanotubes and Gold Nanoparticles for Clinical Applications

This work reports the characterization and application of a new electrochemical platform modified with gold nanoparticles (AuNP) and functionalized multi-walled carbon nanotubes (f-MWCNT) film to determine melatonin (MEL). MEL is a critical sleep inductor neurohormone, categorized by Food and Drug Administration (FDA, USA) as a nutritional supplement. The AuNP were synthesized and stabilized by 3-n-propyl-(4-methylpyridinium) silsesquioxane chloride (Si4Pic+Cl−), presenting an electrostatic stabilization and a material with a positive surface charge. Thermogravimetric analysis (TGA), Fourier transform-infrared spectroscopy (FT-IR), Raman spectroscopy, and zeta (ζ) potential indicated the insertion of oxygenated functional groups and a superficial negative charge for f-MWCNT. The GCE/f-MWCNT-AuNP-Si4Pic+Cl− platform showed an electrocatalytic profile and a current increase of ca. 7.5 times. Voltammograms were obtained by square wave voltammetry (SWV); the current increased with successive additions of MEL in the linear range of 4.9 to 55.5 μmol l−1. The values of LOD and LOQ were 1.6 and 4.7 μmol l−1, respectively. The standard addition was used to quantify MEL in simulated saliva, blood serum, and urine samples. Statistical tests showed no significant differences between the data collected by voltammetry and the comparative technique, molecular absorption spectrometry. It can be concluded that the new method provides novel perspectives for MEL sensing.

Optimization of medium composition for production of a simulated urine sample containing Enterococcus faecalis

Optimization of medium composition for production of a simulated urine sample containing Enterococcus faecalis

Use and quality of point-of-care microscopy, urine culture and susceptibility testing for urinalysis in general practice

Objective: To describe the use and quality of point-of-care (POC) microscopy, urine culture and susceptibility testing performed in general practice in Northern Denmark from 2013 to 2018. Design: Descriptive study Setting: General practices receiving a fee for examining urine samples. Subjects: Simulated urine samples containing uropathogenic bacteria distributed by the organisation for improvement of microbiological quality (MIKAP). Main outcome measures: Percentage of use and correct answers for microscopy, culture and susceptibility testing. Results: A total of 5361 samples were analysed by the use of microscopy (39.7%), culture (66.0%) and/or susceptibility testing (76.5%). For culture, Flexicult SSI urinary kittm (87.6%) demonstrated the highest percentage of correct answers followed by chromogenic agar (85.1%) and 2-plate dipslide (85.2%). Mueller Hinton agar with tablets had the highest percentage of correct answers for susceptibility testing of most bacterial strains (84.6%), followed by Flexicult (77.2%). Furthermore, susceptibility testing with tablets (range: 76.1–84.6%) was found to be more accurate than discs (range: 72.9–75.5%). Overall, the highest percentage of correct answers was obtained when examining urine samples containing Escherichia coli: Microscopy (78.3%), culture (87.0%) and susceptibility testing (range: 84.3–90.7%). Conclusion: The quality of POC testing in general practice was high when examining urine samples containing the most common uropathogen E. coli. Surprisingly, susceptibility testing was more frequently used than culture. This approach may compromise the treatment decision as only cultures contribute with information about the flora composition and bacterial quantification. Interestingly, microscopy was the least used method even though the result may be reached within a few minutes. Key points The quality of POC tests (microscopy, urine culture, susceptibility testing) performed in general practice was high when examining urine containing E. coli, whereas difficulties were observed for samples including S. saprophyticus or K. pneumoniae. Susceptibility testing was more often performed than urine culture, which indicates a problem as only urine cultures contribute with information about the flora composition and bacterial quantification.

From green biowaste to water treatment applications: Utilization of modified new biochar for the efficient removal of ciprofloxacin

Water pollution caused by antibiotics is a serious environmental problem in recent years. Using biochar to remove such pharmaceutical pollutants has recently emerged as a promising option. After H3PO4 modification, a new waste-based biochar (MPCWSB500) from sour cherry stalk was successfully synthesized to remove ciprofloxacin (CFX) from aquatic media, and modification of feedstock has significantly improved the adsorption capacity of biochar. MPCWSB500 is suitable for both batch and continuous treatment systems. The CFX sorption was systematically studied using various kinetics and isotherm models. The surface characteristics of the modified biochar and the possible CFX−biochar interactions were investigated by BET, FT−IR, and SEM−EDX analysis. Short operation time, high sorption capacity (410.06 mg g−1), and nearly 100% removal efficiency were recorded as significant findings at optimum experimental conditions (pH: 6.3, contact time: 40 min, MPCWSB500 dose: 15 mg). Furthermore, the modified biochar exhibited more than 95% CFX removal efficiency in continuous mode at all flow rates (1–10 mL min−1). Its sorption performance was minimally affected by the presence of Cl−, K+, Na+, and NO3− ions in the adsorption medium. In addition, up to 5 sorption-desorption cycles, biochar regeneration and recycling produced satisfactory results. The proposed biochar was also successfully used to remove CFX from simulated hospital wastewater and synthetic urine samples. These features are all important advantages for its real applications. Overall, our research offers a practical approach for removing CFX from the polluted aquatic environment.

Exploration of simulated urine sample biochemistry for the diagnosis of diseases: A laboratory practical exercise

The practical work described here is designed for third-year bachelor students in Life Sciences attending a kidney physiology course. It illustrates how urinary biochemistry can be used for a medical diagnosis. Students have to measure glucose, proteins, and creatinine concentrations in three simulated urine samples. First, they independently elaborate detailed protocols from the biochemical kit's technical sheets. Second, after correction of the protocols by the teacher, they perform the biochemical assays. Finally, students write a report in which they interpret the biochemical data and use them in the context of histological images assigned to each urine sample. With their results, their theoretical background and scientific articles supplied by the teacher, they establish the diagnosis indicating which patients are diseased and from which disease they are suffering (diabetes without nephropathy, diabetic nephropathy or hypertensive nephropathy). The originality of this practical work is to give student autonomy from the start of the project by requiring them to write the protocols, and determine the diagnosis by themselves. Simple and inexpensive, this practical forces students to mobilize their knowledge in renal physiology and pathophysiology as well as those acquired in other disciplines such as biochemistry or cellular biology. A survey conducted on two classes of students confirmed that this type of training helps students to better understand renal physiology, to realize the importance of interdisciplinarity (biochemistry, histology, and physiology) and to develop confidence in their ability to work independently.

The Direct Semi-Quantitative Detection of 18 Pathogens and Simultaneous Screening for Nine Resistance Genes in Clinical Urine Samples by a High-Throughput Multiplex Genetic Detection System.

BackgroundUrinary tract infections (UTIs) are one the most common infections. The rapid and accurate identification of uropathogens, and the determination of antimicrobial susceptibility, are essential aspects of the management of UTIs. However, existing detection methods are associated with certain limitations. In this study, a new urinary tract infection high-throughput multiplex genetic detection system (UTI-HMGS) was developed for the semi-quantitative detection of 18 pathogens and the simultaneously screening of nine resistance genes directly from the clinical urine sample within 4 hours.MethodsWe designed and optimized a multiplex polymerase chain reaction (PCR) involving fluorescent dye-labeled specific primers to detect 18 pathogens and nine resistance genes. The specificity of the UTI-HMGS was tested using standard strains or plasmids for each gene target. The sensitivity of the UTI-HMGS assay was tested by the detection of serial tenfold dilutions of plasmids or simulated positive urine samples. We also collected clinical urine samples and used these to perform urine culture and antimicrobial susceptibility testing (AST). Finally, all urine samples were detected by UTI-HMGS and the results were compared with both urine culture and Sanger sequencing.ResultsUTI-HMGS showed high levels of sensitivity and specificity for the detection of uropathogens when compared with culture and sequencing. In addition, ten species of bacteria and three species of fungi were detected semi-quantitatively to allow accurate discrimination of significant bacteriuria and candiduria. The sensitivity of the UTI-HMGS for the all the target genes could reach 50 copies per reaction. In total, 531 urine samples were collected and analyzed by UTI-HMGS, which exhibited high levels of sensitivity and specificity for the detection of uropathogens and resistance genes when compared with Sanger sequencing. The results from UTI-HMGS showed that the detection rates of 15 pathogens were significantly higher (P<0.05) than that of the culture method. In addition, there were 41(7.72%, 41/531) urine samples were positive for difficult-to-culture pathogens, which were missed detected by routine culture method.ConclusionsUTI-HMGS proved to be an efficient method for the direct semi-quantitative detection of 18 uropathogens and the simultaneously screening of nine antibiotic resistance genes in urine samples. The UTI-HMGS could represent an alternative method for the clinical detection and monitoring of antibiotic resistance.

Rapid, user-friendly, and inexpensive detection of azidothymidine.

Strict adherence to highly active antiretroviral therapy (HAART) is very important to improve the quality of life for HIV-positive patients to reduce new infections and determine treatment success. Azidothymidine (AZT) is an antiretroviral drug commonly used in HAART treatment. In this research, an "add, mix, and measure" assay was developed to detect AZT within minutes. Three different probes designed to release fluorophores when samples containing AZT are added were synthesized and characterized. The limit of detection to AZT in simulated urine samples was determined to be 4μM in 5min for one of the probes. This simple and rapid point-of-care test could potentially be used by clinicians and health care workers to monitor the presence of AZT in low resource settings.

Towards on-Site Detection of Cadmium in Human Urine

Cadmium (Cd) is a non-essential toxic heavy metal. Human exposure to Cd occurs from multiple sources, including diet, tobacco smoke, fossil fuel combustion, and variously contaminated environments. This work reports a rapid method for Cd 2+ detection in simulated urine samples containing glucose (GLC) and actual human urine using the electrochemical technique of square wave anodic stripping voltammetry (ASV). Electrochemical techniques for onsite analysis and detection are preferred for their quick response time, application simplicity, inexpensive instrumentation, and potential portability. This approach is a step forward towards Cd 2+ detection in biological fluids, despite their composition complexity due to possible interference of its constituents. Application of a simple, well controlled, and uniform carbon nanotube (CNT) thin film generated through spinnable CNT arrays enabled us to increase the surface area of traditional glassy carbon electrodes and made possible the detection of nanomolar concentration of Cd ions in urine samples. Employing 120s deposition time on anodic stripping voltammetry led to 1.9 nM and 324 nM limit of detection (LOD) in simulated urine and human samples, respectively. The developed method would facilitate high throughput screening of human urine samples for assessing Cd exposure in future studies.