Normal Human Urine Research Articles

Large-scale identification of calcium oxalate stone inhibitory proteins in normal human urine

Recent evidence has shown that proteins in normal human urine can inhibit calcium oxalate (CaOx) kidney stone formation. Herein, we performed fast protein liquid chromatography (FPLC) to fractionate normal human urinary proteins using anion-exchange (DEAE) and size-exclusion (Superdex 200) materials. FPLC fractions (F1-F15) were examined by CaOx crystallization, growth, aggregation and crystal-cell adhesion assays. The fractions with potent inhibitory activities against CaOx crystals were then subjected to mass spectrometric protein identification. The data revealed that 13 of 15 fractions showed inhibitory activities in at least one crystal assay. Integrating CaOx inhibitory scores demonstrated that F6, F7 and F8 had the most potent inhibitory activities. NanoLC-ESI-Qq-TOF MS/MS identified 105, 93 and 53 proteins in F6, F7 and F8, respectively. Among them, 60 were found in at least two fractions and/or listed among known inhibitors with solid experimental evidence in the StoneMod database (https://www.stonemod.org). Interestingly, 10 of these 60 potential inhibitors have been reported with lower urinary levels in CaOx stone formers compared with healthy (non-stone) individuals, strengthening their roles as potent CaOx stone inhibitors. Our study provides the largest dataset of potential CaOx stone inhibitory proteins that will be useful for further elucidations of stone-forming mechanisms and ultimately for therapeutic/preventive applications.

Microwave Characterization and Assessment of the Dielectric Properties of Normal Human Urine

Microwave Characterization and Assessment of the Dielectric Properties of Normal Human Urine

Linker-Preserved Iron Metal-Organic Framework-Based Lateral Flow Assay for Sensitive Transglutaminase 2 Detection in Urine Through Machine Learning-Assisted Colorimetric Analysis.

A groundbreaking demonstration of the utilization of the metal-organic framework MIL-101(Fe) as an exceptionally perceptive visual label in colorimetric lateral flow assays (LFA) is described. This pioneering approach enables the precise identification of transglutaminase 2 (TGM2), a recognized biomarker for chronic kidney disease (CKD), in urine specimens, which offers a remarkably sensitive naked-eye detection mechanism. The surface of MIL-101(Fe) was modified with oxalyl chloride, adipoyl chloride, and poly(acrylic) acid (PAA); these not only improved the labeling material stability in a complex matrix but also achieved a systematic control in the detection limit of the TGM2 concentration using our LFA platform. The advanced LFA with the MIL-101(Fe)-PAA label can detect TGM2 concentrations down to 0.012, 0.009, and 0.010 nM in Tris-HCl buffer, urine, and desalted urine, respectively, which are approximately 55-fold lower than those for a conventional AuNP-based LFAs. Aside from rapid TGM2 detection (i.e., within 20 min), the performance of the MIL-101(Fe)-PAA-based LFA on reproducibility [coefficients of variation (CV) < 2.9%] and recovery (95.9-103.2%) along with storage stability within 25 days of observation (CV < 6.0%) shows an acceptable parameter range for quantitative analysis. A sophisticated sensing method grounded in machine learning principles was also developed, specifically aimed at precisely deducing the TGM2 concentration by analyzing immunoreaction sites. More importantly, our developed LFA offers potential for clinical measurement of TGM2 concentration in normal human urine and CKD patients' samples.

The modulatory effects of large and small extracellular vesicles from normal human urine on calcium oxalate crystallization, growth, aggregation, adhesion on renal cells, and invasion through extracellular matrix: An in vitro study

Urinary extracellular vesicles (uEVs) play important roles in physiologic condition and various renal/urological disorders. However, their roles in kidney stone disease remain unclear. This study aimed to examine modulatory effects of large and small uEVs derived from normal human urine on calcium oxalate (CaOx) crystals (the main component in kidney stones). After isolation, large uEVs, small uEVs and total urinary proteins (TUPs) with equal (protein equivalent) concentration were added into various crystal assays to compare with the control (without uEVs or TUPs). TUPs strongly inhibited CaOx crystallization, growth, aggregation and crystal-cell adhesion. Large uEVs had lesser degree of inhibition against crystallization, growth and crystal-cell adhesion, and comparable degree of aggregation inhibition compared with TUPs. Small uEVs had comparable inhibitory effects as of TUPs for all these crystal assays. However, TUPs and large uEVs slightly promoted CaOx invasion through extracellular matrix, whereas small uEVs did not affect this. Matching of the proteins reported in six uEVs datasets with those in the kidney stone modulator (StoneMod) database revealed that uEVs contained 18 known CaOx stone modulators (mainly inhibitors). These findings suggest that uEVs derived from normal human urine serve as CaOx stone inhibitors to prevent healthy individuals from kidney stone formation.

Development of a point-of-care colorimetric metabolomic sensor platform

Metabolomics is the large-scale study of small molecule metabolites within a biological system. It has applications in measuring dietary intake, predicting heart disease risk, and diagnosing cancer. Metabolites are often measured using high-end analytical tools such as mass spectrometers or large spectrophotometers. However, due to their size, cost, and need for skilled operators, using such equipment at the bedside is not practical. To address this issue, we have developed a low-cost, portable, optical color sensor platform for metabolite detection. This platform includes LEDs, sensors, microcontrollers, a power source, and a Bluetooth chip enclosed within a 3D-printed light-tight case. We evaluated the color sensor's performance using both a range of dyed water samples as well as well-established colorimetric reactions for specific metabolite detection. The sensor accurately measured creatinine, L-carnitine, ascorbate, and succinate well within normal human urine levels with accuracy and sensitivity equal to or better than a standard laboratory spectrophotometer. Our color sensor offers a cost-effective, portable alternative for measuring metabolites via colorimetric assays, thereby enabling low-cost, point-of-care metabolite testing.

An improved method for measuring catalase activity in biological samples.

Catalase (CAT) is an important enzyme that protects biomolecules against oxidative damage by breaking down hydrogen peroxide (H2O2) into water and oxygen. CAT is present in all aerobic microbes, animals, and plants. It is, however, absent from normal human urine but can be detected in pathological urine. CAT testing can thus help to detect such urine. This study presents a novel spectrophotometric method for determining CAT activity characterized by its simplicity, sensitivity, specificity, and rapidity. The method involves incubating enzyme-containing samples with a carefully chosen concentration of H2O2 for a specified incubation period. Subsequently, a solution containing ferrous ammonium sulfate (FAS) and sulfosalicylic acid (SSA) is added to terminate the enzyme activity. A distinctive maroon-colored ferrisulfosalicylate complex is formed. The formation of this complex is a direct result of the reaction between FAS and any residual peroxide present. This leads to the generation of ferric ions when coordinated with SSA. The complex has a maximum absorbance of 490 nm. This advanced method eliminates the need for concentrated acids to stop CAT activity, making it safer and easier to handle. A comparative analysis against the standard ferrithiocyanate method showed a correlation coefficient of 0.99, demonstrating the new method's comparable effectiveness and reliability. In conclusion, a simple and reliable protocol for assessing CAT activity, which utilizes a cuvette or microplate, has been demonstrated in this study. This interference-free protocol can easily be used in research and clinical analysis with considerable accuracy and precision.

Uromodulin in Kidney Health and Disease: Genetic Variants, Biomarker Applications, and Clinical Implications

Uromodulin, also known as Tamm-Horsfall protein, is the most abundant protein in normal human urine and plays a crucial role in kidney function. This review explores the multifaceted role of uromodulin in renal physiology and pathophysiology, focusing on its genetic basis and utility as a biomarker. We systematically reviewed literature published between 2000 and 2024, analyzing studies on uromodulin's biomarker potential and genetic associations with kidney diseases. Our findings reveal that uromodulin is a promising biomarker for various renal conditions, including chronic kidney disease, acute kidney injury, and urinary tract infections. Urinary uromodulin levels correlate with kidney function and can predict disease progression and outcomes. Additionally, we examined genetic variants in the UMOD gene and their associations with kidney diseases. Several Single Nucleotide Polymorphisms (SNPs) in UMOD have been linked to an increased risk of chronic kidney disease, kidney stones, and hypertension. The interplay between genetic variants and uromodulin's biomarker capabilities offers potential for personalized medicine approaches in nephrology. However, standardization of uromodulin measurement techniques and more extensive prospective studies are needed to establish its clinical utility fully. This review synthesizes current knowledge on uromodulin's dual role as a genetically influenced protein and a renal biomarker, providing insights for future research directions and potential clinical applications in kidney disease management.

Assessment ofurinary 6-hydroxy-2,4-cyclohexadienyl mercapturic acid as a novel biomarker ofbenzene exposure.

Assessing benzene exposure is a public health priority due to its deleterious health effects and ubiquitous industrial and environmental sources of exposure. Phenyl mercapturic acid (PhMA) is a commonly used urinary biomarker to assess benzene exposure. However, recent work has identified significant interlaboratory variation in urinary PhMA concentrations related to methodological differences. In this study, we present urinary 6-hydroxy-2,4-cyclohexadienyl mercapturic acid (pre-PhMA), a metabolite that undergoes acid-catalyzed dehydration to form PhMA, as a novel and specific urinary biomarker for assessing benzene exposure. We developed and validated the first quantitative liquid chromatography-tandem mass spectrometry assay for measuring urinary concentrations of pre-PhMA. The pH effect on the method of ruggedness testing determined that pre-PhMA is stable across the normal human urine pH range and that neutral conditions must be maintained throughout quantification for robust and accurate measurement of urinary pre-PhMA concentrations. The method exhibited below 2 ng/mL sensitivity for pre-PhMA, linearity over three orders of magnitude, and precision and accuracy within 10%. Urinary pre-PhMA concentrations were assessed in 369 human urine samples. Smoking individuals exhibited elevated levels of pre-PhMA compared to non-smoking individuals. Furthermore, the relationship between benzene exposure and urinary pre-PhMA levels was explored by examining the correlation of pre-PhMA with 2-cyanoethyl mercapturic acid, a smoke exposure biomarker. The urinary biomarkers exhibited a positive correlation (r = 0.720), indicating that pre-PhMA levels increased with benzene exposure. The results of this study demonstrate that urinary pre-PhMA is a rugged and effective novel biomarker of benzene exposure that can be widely implemented for future biomonitoring studies.

Ratio-Metric Fluorescence/Colorimetric and Smartphone-Assisted Visualization for the Detection of Dopamine Based on Cu-MOF with Catecholase-like Activity

As the most abundant catecholamine neurotransmitter in the brain, dopamine plays an important role in the normal physiological process, and its level in urine also changes during human pathological processes. In clinic, the detection of dopamine in urine is a potential marker for the diagnosis and the treatment of endocrine-related diseases. In this work, a copper metal organic framework with catecholase-like activity was prepared via the precipitation of Cu2+ and imidazole, simulating the N-Cu coordination environment in the active site of catecholase. Cu-MOF (the copper–metal organic framework) can catalyze the oxidation of DA (dopamine) to dopaquinone using O2 in the air. The oxidation product can further react with 1,3-dihydroxynaphthalene to produce a fluorophore product. Based on the above reaction, a multimodal sensing platform with three signal outputs, including ratio-metric fluorescence, absorbance and digital information extracted from smartphone images for simple and sensitive determination of DA, was proposed, with detection limits of 0.0679, 0.3206, and 0.3718 μM, respectively. This multimodal sensing platform was able to detect DA in body fluid in a self-correcting way, as demonstrated by the successful determination of DA in normal human urine samples, and samples with a high level of interference.

Epidermal Growth factor in human urine as promotor for the growth of Leishmania sp. In vitro.

Leishmania parasites are the causal agents of leishmaniasis, a group of protozoan diseases transmitted to mammals, including human beings, by phlebotomine sandflies. In culture media (at 25-28̊C ,pH =7.2-7.4),leishmania parasites develop as motile promastigotes similar to those found in the sand fly midgut . Traditionally media available do not meet the requirement for the bulk cultivation of Leishmania parasites ,it requires fetal calf serum (FCS),that is very expensive and not easily available in the market. A number of studies have shown that the addition of 5-10 % normal human urine stimulates growth, leading to more rapid multiplication and a higher concentration of parasites .Urine from patients with bladder cancer were used in this study to determine the effect of Epidermal growth factor ( which is increased in level in this type of urine),and our study showed that proliferation indexes were significantly increased in the culture media supplemented with human urine from patients with bladder cancer, we undertook a detailed study of such an effect in old world Leishmania isolates causing cutaneous Leishmaniasis. We also found that urine with high percentage of EGF. Could be used as an alternative of fetal calf serum.

Contamination of bacterial extracellular vesicles (bEVs) in human urinary extracellular vesicles (uEVs) samples and their effects on uEVs study

AbstractBacterial overgrowth is common for improperly stored urine. However, its effects on human urinary extracellular vesicles (uEVs) study had not been previously examined nor documented. This study investigated the presence of bacterial EVs (bEVs) contaminated in uEVs samples and their effects on uEVs study. Nanoscale uEVs were isolated from normal human urine immediately after collection (0‐h) or after 25°C‐storage with/without preservative (10 mM NaN3) for up to 24‐h. Turbidity, bacterial count and total uEVs proteins abnormally increased in the 8‐h and 24‐h‐stored urine without NaN3. NanoLC‐ESI‐LTQ‐Orbitrap MS/MS identified 6–13 bacterial proteins in these contaminated uEVs samples. PCR also detected bacterial DNAs in these contaminated uEVs samples. Besides, uEVs derived from 8‐h and 24‐h urine without NaN3 induced macrophage activation (CD11b and phagocytosis) and secretion of cytokines (IFN‐α, IL‐8, and TGF‐β) from macrophages and renal cells (HEK‐293, HK‐2, and MDCK). All of these effects induced by bacterial contamination were partially/completely prevented by NaN3. Interestingly, macrophage activation and cytokine secretion were also induced by bEVs purified from Escherichia coli. This study clearly shows evidence of bEVs contamination and their effects on human uEVs study when the urine samples were inappropriately stored, whereas NaN3 can partially/completely prevent such effects from the contaminated bEVs.

A strategy of “fluorescence-visualization dual channel” for highly sensitive and rapid detection of phosphate biomarkers based on stimulus-responsive infinite coordination polymers

The novel stimulus-responsive host-guest nanoparticles were prepared based on lanthanide infinite coordination polymer ([email protected]) for rapid and point-of-care analysis of phosphate (Pi) biomarkers. The obtained [email protected], which have the host-guest structure, were applied to high-sensitive detection of Pi biomarkers by the means of ratiometric “fluorescence-visualization dual channel”. The established method has the advantages of low detection limit (51 nM), wide linear range (0.25–5 μM) and rapid response to Pi within 3 min. The developed sensor was used to detect Pi in normal human urine and serum samples, with the recoveries of 96.04–106.3%. Furthermore, the fluorescent color changes of [email protected] from blue to red can be used to visually inspect Pi by the naked eyes. More importantly, a portable test strip based on [email protected] was fabricated and successfully applied to the rapid visual detection of Pi in human urine and serum samples.

A Comprehensive 2D-LC/MS/MS Profile of the Normal Human Urinary Metabolome.

Profiling bodily fluids is crucial for monitoring and discovering metabolic markers of disease. In this study, a comprehensive analysis approach based on 1D-LC-MS/MS and 2D-LC-MS/MS was applied to profile normal human urine metabolites from 348 children and 315 adults. A total of 2357 metabolites were identified, including 1831 endogenous metabolites and 526 exogenous ones. In total, 1005 metabolites were identified in urine for the first time. The urinary metabolites were mainly involved in amino acid metabolism, small molecule biochemistry, lipid metabolism and cellular compromise. The comparison of adult’s and children’s urine metabolomes showed adults urine had more metabolites involved in immune response than children’s, but the function of binding of melatonin, which belongs to the endocrine system, showed a higher expression in children. The urine metabolites detected by the 1D-LC-MS/MS method were mainly related to amino acid metabolism and lipid metabolism, and the 2D-LC-MS/MS method not only explored metabolites from 1D-LC-MS/MS but also metabolites related to cell signaling, cell function and maintenance, etc. Our analysis comprehensively profiled and functionally annotated the metabolome of normal human urine, which would benefit the application of urinary metabolome to clinical research.

Development of the certified reference material of chromium in freeze-dried human urine

To develop the certified reference material in freeze-dried human urine. Normal human urine was collected, mixed and filtered as a matrix, then the urine was prepared by adding standard, mixing, sub-packaging, and freeze-drying; After the homogeneity and stability were tested by inductively coupled plasma mass spectrometer, the urine were determined by two methods in seven independent laboratories and the uncertainty of the certified reference material was evaluated. The minimum sample intake of the developed certified reference material was 0.50 mL, and the homogeneity was good. The freeze-dried material was stable for 12 months at ≤4 ℃, and the reference material was stable at 4 ℃ after reconstitution for the 7 d. The standard value and uncertainty of the two levels were(16.0±1.0) μg/L and(30.7±2.1) μg/L(k=2), respectively. The certified reference material of chromium in freeze-dried human urine in this research met the national certified reference material requirements and could be used for the quality control of the detection of chromium in laboratory human urine.

Comparative and Selective Interaction of Amino Acid d-Cysteine with Colloidal Gold Nanoparticles in the Presence of a Fluorescent Probe in Aqueous Medium.

In this communication, we report the comparative and selective interaction of amino acid d-cysteine (d-Cys) with citrate caped gold nanoparticles (Au NPs) in the presence of a fluorescent dye, rhodamine B (RhB), in aqueous solution. Au NPs of size 27.5 nm could almost fully quench the steady-state fluorescence emission of RhB at their optimum concentrations in the mixed solution. The interactions of d-Cys, l-Cys, all other relevant d- and l-amino acids, neurotransmitters, and other relevant biological compounds with the Au NPs/RhB mixed solution have been explored by monitoring the fluorescence recovery efficiencies from the almost fully quenched state of RhB fluorescence via a simple steady-state spectrofluorometric method. The higher fluorescence recovery for the interaction of d-Cys with the Au NPs/RhB mixed system is accompanied by a distinct color change (red-wine to bluish-black) of the assay medium after the reaction compared to that of all other interfering compounds considered in this work. The sensitivity of this fluorometric response lies in a broad linear range of concentrations of d-Cys and the limit of detection (LOD) is found to be 4.2 nM, which is low compared to many other methods available in the literature. The different degrees of interaction of d-Cys and l-Cys with the Au NPs/RhB mixed sample have been further explored by circular dichroism (CD) spectroscopy and Fourier transform infrared (FTIR) spectroscopy. The selective interaction of d-Cys with the proposed Au NPs/RhB mixed system is also found to be correlated with interparticle cross-linking and aggregations of nanoparticles by the analysis of ζ potential and dynamic light scattering (DLS) study, transmission electron microscopy (TEM), atomic force microscopy (AFM), UV–vis absorption spectroscopy etc. The proposed interaction mechanism is further studied with a normal human urine sample to elucidate that the optimized combination of Au NPs and RhB may be realized as an efficient platform for detection of the amino acid d-Cys in a real biosample via a simple fluorometric approach.

The assistant effects of porcine fibrin sealant in improving stone clearance rate in flexible ureteroscopy lithotripsy in ex vivo porcine kidney model

Objectives: To investigate the feasibility of using a porcine fibrin sealant to wrap and remove kidney calculi fragments through an isolated porcine kidney model. Methods: In the isolated porcine kidney stone model (implanted with 100 mg, air dried, ≤1 mm human stone fragments, n=6;implanted with 100 mg, air dried, ≤3 mm human stone fragments, n=6), the ureteral soft mirror combined with the 12/14Fr UAS was used to test the effect of stone extraction using only two stone extraction methods: basket extraction (control group, ≤1 mm stone fragments, n=3; ≤3 mm stone fragments, n=3) and basket-sealant extraction (test group, ≤1 mm stone fragments, n=3; ≤3 mm stone fragments, n=3). Compare the stone removal rate and operation time of the two stone retrieval methods. The sealant was put into urine of normal human and observed. Results: Porcine Fibrin Sealant can form a gel in saline and urine and adhere and wrap stone fragments. The time of procedures of test (basket-sealant) and control (basket) group in kidneys implanted with ≤ 1 mm stone fragments were (14.0±4.2) and (29.0±0.7)min (P<0.05) stone clearance rates were (90.9±1.4)% and (48.4±15.7)% (P<0.05), respectively. In kidneys implanted with ≤ 3 mm fragments, time of procedures were (12.8±4.0) and (30.0±0)min (P<0.05) Stone clearance rates were (91.1±5.0)% and (20.7±8.0)% (P<0.05). The Sealant dissolves by itself in normal human urine and normal saline at 37 ℃ for 24 hours. Conclusion: The appropriate concentration of Porcine Fibrin Sealant assisted stone retrieval may become a new method for removing small stone fragments in retrograde intrarenal surgery.

Synergistic inhibition of the urinary tract pathogen Staphylococcus saprophyticus by curcumin and berberine

Staphylococcus saprophyticus is a Gram-positive pathogen that is most commonly associated with community-acquired urinary tract infections in adolescent and adult women. While antibiotics can be used to treat these infections, they often reoccur and there has been increased interest in using herbal preparations to treat them. Curcumin has been promoted as a treatment for a variety of diseases, but its effectiveness is limited by its low solubility in aqueous solutions and poor bioavailability. The sensitivity of S. saprophyticus to curcumin and berberine was determined by plate count assays, minimum inhibitory concentrations in microtiter plates, and growth kinetics. When 100 µg ml-1 berberine was combined with 20 µg ml-1 curcumin, there was a large increase in the lag phase and a decrease in the growth rate and final yield of bacteria. There was complete inhibition of growth in normal human urine. These studies indicate that a combination of curcumin with berberine allows curcumin to be used at much lower concentrations.

A Zn‐based metal–organic framework for the irreversible determination of trace biomarkers of styrene and ethylbenzene in urine

AbstractPhenylglyoxylic acid (PGA), as a urine metabolite of ethylbenzene and styrene (EB/S), can effectively represent the level of EB/S ingested by the human body through oral inhalation and skin absorption. Compared with measuring EB/S levels directly in the environment, detecting the level of PGA in urine has the advantages of high specificity and noninvasive sampling. In this paper, a metal–organic framework (MOF)‐based sensorZn‐PBA = ({[Zn(PBA)·H2O]·2H2O}n, H2PBA = 4‐(5‐pyridinyl)‐1,3 benzoic acid) for trace detection of PGA was designed and synthesized by a solvothermal method.Zn‐PBAcould detect the level of PGA in normal human physiological urine environment (pH range from 5.5 to 7.5) and has the characteristics of high selectivity and sensitivity, rapid response (&lt;210 s), low limit of detection (LOD = 1.96 × 10−6 M), and so forth. The irreversible sensing of PGA proves that PGA caused the structural collapse ofZn‐PBA. This paper provides a specific sensor for detecting trace PGA level for evaluating the EB/S content intake by the human body.

Reduction of susceptibility to azoles and 5-fluorocytosine and growth acceleration in Candida albicans in glucosuria

Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia and glucosuria, and is a risk factor for Candida infections. To reveal the potential effects of glucosuria on Candida spp., we investigated their growth and antifungal susceptibilities in normal human urine to which glucose was added. The viable cell numbers of Candida spp. were more than 10 fold higher in the urine added 3000 mg/dL glucose than in plain urine. In antifungal susceptibility, more than 80% of Candida albicans clinical isolates increased minimum inhibitory concentrations of azoles and 5-fluorocytosine with the addition of glucose, and exceeded their breakpoints. In most of the C. albicans clinical isolates, the mRNA expression of the azole resistance genes ERG11, CDR1, CDR2, and MDR1 in the presence of glucose in urine. These observations provide valuable information about the clinical course and therapeutic effects of azoles against C. albicans infections in patients with diabetes mellitus and hyperglucosuria.

Highly sensitive non-enzymatic glucose sensor based on carbon nanotube microelectrode set

In this work, a carbon nanotube microelectrode set (CNT µ-ES) was modified, in which three electrodes based on highly densified carbon nanotube fiber (HD-CNTf) cross-sections (length ~40 µm) were embedded in an inert polymer matrix with exposed open-ended CNTs at the interface. An HD-CNTf cross-section (∼40 µm diameter) electrochemically modified with copper nanoparticles (CuNPs) was used as the working electrode; a bare HD-CNTf cross-section (∼94 µm diameter) was used as the counter electrode; and an HD-CNTf cross-section (∼94 µm diameter) electroplated with Ag/AgCl and then coated with Nafion™ was used as a quasi-reference electrode. The electrochemical activity of the CuNPs/HD-CNTf microsensor for glucose electrooxidation was examined by cyclic voltammetry and amperometry in 0.1 M NaOH solution. As shown by amperometry studies, the proposed nonenzymatic CuNPs/HD-CNTf microsensor had a remarkably low limit of detection (28 nM) and a wide linear quantification range with an excellent sensitivity (1942 nA·µM−1·cm−2). This sensitivity can be attributed to the synergetic effect of electrocatalytic CuNPs and aligned HD-CNTf, which provide excellent conductivity. The electrooxidation of glucose on the developed microsensor was free from chloride poisoning and independent of the oxygen concentration. The developed microsensor demonstrated insignificant interference from the oxidation of common interfering species and carbohydrate compounds at their physiological concentrations. Finally, the CuNPs/ HD-CNTf microsensor was successfully applied for the quantification of glucose in normal human serum and diabetic patient urine samples, demonstrating the applicability of this strategy for commercial nonenzymatic glucose sensors.