ABSTRACT
Screen-printed carbon electrodes (SPCE) functionalized with MXene-based three-dimensional nanomaterials are reported for rapid determination of creatinine. Ti3C2TX MXene with in situ reduced AuNPs (MXene@AuNP) were used as a coreactant accelerator for efficient immobilization of enzymes. Creatinine could be oxidized by chitosan-embedded creatinine amidohydrolase, creatine amidinohydrolase, or sarcosine oxidase to generate H2O2, which could be electrochemically detected enhanced by Prussian blue (PB). The enzyme@CS/PB/MXene@AuNP/SPCE detected creatinine within the range 0.03-4.0 mM, with a limit of detection of 0.01 mM, with an average recovery of 96.8-103.7%. This indicates that the proposed biosensor is capable of detecting creatinine in a short amount of time (4 min) within a ± 5% percentage error, in contrast with the standard clinical colorimetric method. With this approach, reproducible and stable electrochemical responses could be achieved for determination of creatinine in serum, urine, or saliva. These results demonstrated its potential for deployment in resource-limited settings for early diagnosis and tracking the progression of chronic kidney disease (CKD).
Subject(s)
Biosensing Techniques , Carbon , Creatinine , Electrochemical Techniques , Electrodes , Ferrocyanides , Gold , Hydrogen Peroxide , Limit of Detection , Metal Nanoparticles , Sarcosine Oxidase , Ureohydrolases , Creatinine/blood , Creatinine/urine , Carbon/chemistry , Humans , Sarcosine Oxidase/chemistry , Gold/chemistry , Biosensing Techniques/methods , Biosensing Techniques/instrumentation , Ferrocyanides/chemistry , Electrochemical Techniques/methods , Electrochemical Techniques/instrumentation , Hydrogen Peroxide/chemistry , Metal Nanoparticles/chemistry , Ureohydrolases/chemistry , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Chitosan/chemistry , Point-of-Care Testing , Amidohydrolases , TitaniumABSTRACT
The diagnosis of disease biomarkers is crucial for the identification, monitoring, and prognostic assessment of malignant disease. However, biological samples with autofluorescence, complex components, and heterogeneity pose major challenges to reliable biosensing. Here, we report the self-assembly of natural proteins and the triplet-triplet annihilation upconversion (TTA-UC) pair to form upconverted protein clusters (â¼8.2 ± 1.1 nm), which were further assembled into photon upconversion supramolecular assemblies (PUSA). This PUSA exhibited unique features, including a small size (â¼44.1 ± 4.1 nm), oxygen tolerance, superior biocompatibility, and easy storage via lyophilization, all of which are long sought after for photon upconversion materials. Further, we have revealed that the steric hindrance of the annihilator suppresses the stacking of the annihilator in PUSA, which is vital for maintaining the water dispersibility and enhancing the upconversion performance of PUSA. In conjunction with sarcosine oxidase, this near infrared (NIR)-excitable PUSA nanoprobe could perform background-free biosensing of urinary sarcosine, which is a common biomarker for prostatic carcinoma (PCa). More importantly, this nanoprobe not only allows for qualitative identification of urinary samples from PCa patients by the unaided eye under NIR-light-emitting diode (LED) illumination but also quantifies the concentration of urinary sarcosine. These remarkable findings have propelled photon upconversion materials to a new evolutionary stage and expedited the progress of upconversion biosensing in clinical diagnostics.
Subject(s)
Biosensing Techniques , Photons , Humans , Sarcosine/urine , Sarcosine/chemistry , Sarcosine Oxidase/chemistry , Proteins/analysis , Proteins/chemistryABSTRACT
BACKGROUND: Early prostatic cancer (PCa) diagnosis significantly improves the chances of successful treatment and enhances patient survival rates. Traditional enzyme cascade-based early cancer detection methods offer efficiency and signal amplification but are limited by cost, complexity, and enzyme dependency, affecting stability and practicality. Meanwhile, sarcosine (Sar) is commonly considered a biomarker for PCa development. It is essential to develop a Sar detection method based on cascade reactions, which should be efficient, low skill requirement, and suitable for on-site testing. RESULTS: To address this, our study introduces the synthesis of organic-inorganic self-assembled nanoflowers to optimize existing detection methods. The Sar oxidase (SOX)-inorganic hybrid nanoflowers (Cu3(PO4)2:Ce@SOX) possess inherent fluorescent properties and excellent peroxidase activity, coupled with efficient enzyme loading. Based on this, we have developed a dual-mode multi-enzyme cascade nanoplatform combining fluorescence and colorimetric methods for the detection of Sar. The encapsulation yield of Cu3(PO4)2:Ce@SOX reaches 84.5 %, exhibiting a remarkable enhancement in catalytic activity by 1.26-1.29 fold compared to free SOX. The present study employing a dual-signal mechanism encompasses 'turn-off' fluorescence signals ranging from 0.5 µM to 60 µM, with a detection limit of 0.226 µM, and 'turn-on' colorimetric signals ranging from 0.18 µM to 60 µM, with a detection limit of 0.120 µM. SIGNIFICANCE: Furthermore, our study developed an intelligent smartphone sensor system utilizing cotton swabs for real-time analysis of Sar without additional instruments. The nano-platform exhibits exceptional repeatability and stability, rendering it well-suited for detecting Sar in authentic human urine samples. This innovation allows for immediate analysis, offering valuable insights for portable and efficient biosensors applicable to Sar and other analytes.
Subject(s)
Colorimetry , Oxidation-Reduction , Sarcosine , Smartphone , Sarcosine/urine , Sarcosine/analysis , Sarcosine/chemistry , Humans , Nanostructures/chemistry , Limit of Detection , Spectrometry, Fluorescence , Prostatic Neoplasms/diagnosis , Fluorescence , Biosensing Techniques , Sarcosine Oxidase/chemistryABSTRACT
CYP116B5 is a class VII P450 in which the heme domain is linked to a FMN and 2Fe2S-binding reductase. Our laboratory has proved that the CYP116B5 heme domain (CYP116B5-hd) is capable of catalyzing the oxidation of substrates using H2O2. Recently, the Molecular Lego approach was applied to join the heme domain of CYP116B5 to sarcosine oxidase (SOX), which provides H2O2 in-situ by the sarcosine oxidation. In this work, the chimeric self-sufficient fusion enzyme CYP116B5-SOX was heterologously expressed, purified, and characterized for its functionality by absorbance and fluorescence spectroscopy. Differential scanning calorimetry (DSC) experiments revealed a TM of 48.4 ± 0.04 and 58.3 ± 0.02°C and a enthalpy value of 175,500 ± 1850 and 120,500 ± 1350 cal mol-1 for the CYP116B5 and SOX domains respectively. The fusion enzyme showed an outstanding chemical stability in presence of up to 200 mM sarcosine or 5 mM H2O2 (4.4 ± 0.8 and 11.0 ± 2.6% heme leakage respectively). Thanks to the in-situ H2O2 generation, an improved kcat/KM for the p-nitrophenol conversion was observed (kcat of 20.1 ± 0.6 min-1 and KM of 0.23 ± 0.03 mM), corresponding to 4 times the kcat/KM of the CYP116B5-hd. The aim of this work is the development of an engineered biocatalyst to be exploited in bioremediation. In order to tackle this challenge, an E. coli strain expressing CYP116B5-SOX was employed to exploit this biocatalyst for the oxidation of the wastewater contaminating-drug tamoxifen. Data show a 12-fold increase in tamoxifen N-oxide production-herein detected for the first time as CYP116B5 metabolite-compared to the direct H2O2 supply, equal to the 25% of the total drug conversion.
Subject(s)
Biodegradation, Environmental , Cytochrome P-450 Enzyme System , Escherichia coli , Hydrogen Peroxide , Sarcosine Oxidase , Hydrogen Peroxide/metabolism , Cytochrome P-450 Enzyme System/metabolism , Cytochrome P-450 Enzyme System/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Sarcosine Oxidase/metabolism , Sarcosine Oxidase/genetics , Sarcosine Oxidase/chemistry , Mixed Function Oxygenases/metabolism , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/chemistry , Oxidation-Reduction , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/chemistry , Sarcosine/metabolism , Sarcosine/analogs & derivativesABSTRACT
N-Methylisothiazolinone (MIT) is a thiol group modifier and antimicrobial agent. Arthrobacter sarcosine oxidase (SoxA), a diagnostic enzyme for assaying creatinine, loses its activity upon the addition of MIT, and its inactivation mechanism remains unclear. In this study, SoxA was chemically modified using MIT (mo-SoxA), and its structural and chemical properties were characterized. Spectral analysis data, oxygen consumption rates, and reactions were compared between intact SoxA and mo-SoxA. These demonstrate that the oxidative half-reaction toward oxygen is inhibited by MIT modification. The oxidase activity of mo-SoxA was approximately 2.1% of that of intact SoxA, and its dehydrogenase activity was approximately 4.2 times higher. The C-to-S mutants revealed that cooperative modification of 2 specific cysteine residues caused a drastic change in the enzyme reaction mode. Based on the modeled tertiary structures, the putative entrance for oxygen uptake is predicted to be blocked by the chemical modification of the 2 cysteine residues.
Subject(s)
Arthrobacter , Oxygen , Sarcosine Oxidase , Thiazoles , Arthrobacter/enzymology , Cysteine/chemistry , Cysteine/metabolism , Kinetics , Models, Molecular , Oxidation-Reduction , Oxygen/metabolism , Oxygen/chemistry , Sarcosine Oxidase/metabolism , Sarcosine Oxidase/chemistry , Sarcosine Oxidase/genetics , Thiazoles/pharmacologyABSTRACT
Efficient biocatalytic cascade reactions play a crucial role in guiding intricate, specific and selective intracellular transformation processes. However, the catalytic activity of the enzyme cascade reaction in bulk solution was greatly impacted by the spatial morphology and inter-enzyme distance. The programmability and addressability nature of framework nucleic acid (FNA) allows to be used as scaffold for immobilization and to direct the spatial arrangement of enzyme cascade molecules. Here, we used tetrahedral DNA framework (TDF) as nanorulers to assemble two enzymes for constructing a double-enzyme complex, which significantly enhance the catalytic efficiency of sarcosine oxidase (SOx)/horseradish peroxidase (HRP) cascade system. We synthesized four types of TDF nanorulers capable of programming the lateral distance between enzymes from 5.67â nm to 12.33â nm. Enzymes were chemical modified by ssDNA while preserving most catalytic activity. Polyacrylamide gel electrophoresis (PAGE), transmission electron microscopy (TEM) and atomic force microscopy (AFM) were used to verify the formation of double-enzyme complex. Four types of double-enzyme complexes with different enzyme distance were constructed, in which TDF26(SOx+HRP) exhibited the highest relative enzyme cascade catalytic activity, ~3.11-fold of free-state enzyme. Importantly, all the double-enzyme complexes demonstrate a substantial improvement in enzyme cascade catalytic activity compared to free enzymes.
Subject(s)
Biocatalysis , DNA , Horseradish Peroxidase , Sarcosine Oxidase , Horseradish Peroxidase/chemistry , Horseradish Peroxidase/metabolism , DNA/chemistry , DNA/metabolism , Sarcosine Oxidase/chemistry , Sarcosine Oxidase/metabolism , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolismABSTRACT
Enzymes are usually stereospecific against chiral substrates, which is commonly accepted for the amine oxidase family of enzymes as well. However, the FsqB (fumisoquin biosynthesis gene B) enzyme that belongs to the family of sarcosine oxidase and oxidizes L-N-methyl-amino acids, shows surprising activity for both enantiomers of N-methyl-dopa. The aim of this study is to understand the mechanism behind this behavior. Primary docking experiments showed that tyrosine and aspartate residues (121 and 315 respectively) are located on the ceiling of the active site of FsqB and may play a role in fixing the N-methyl-dopa via its catechol moiety and allowing both stereoisomers of this substrate to be in close proximity of the N5 atom of the isoalloxazine ring of the cofactor. Three experimental approaches were used to prove this hypothesis which are: (1) studying the oxidative ability of the variants Y121F and D315A on N-methyl-dopa substrates in comparison with N-methyl-tyrosine substrates; (2) studying the FsqB WT and variants catalyzed biotransformation via high-performance liquid chromatography (HPLC); (3) molecular dynamics simulations to characterize the underlying mechanisms of the molecular recognition. First, we found that the chemical characteristics of the catechol moiety of N-methyl-dopa are important to explain the differences between N-methyl-dopa and N-methyl-tyrosine. Furthermore, we found that Y121 and D315 are specific in FsqB and not found in the model enzyme sarcosine oxidase. The on-bench and theoretical mutagenesis studies show that Y121 residue has a major role in fixing the N-methyl-dopa substrates close to the N5 atom of the isoalloxazine ring of the cofactor. Simultaneously, D315 has a supportive role in this mechanism. Jointly, the experimental and theoretical approaches help to solve the riddle of FsqB amine oxidase substrate specificity.
Subject(s)
Aspergillus fumigatus , Fungal Proteins , Sarcosine Oxidase , Stereoisomerism , Azoles , Drug Resistance, Fungal , Tyrosine , Methyldopa , KineticsABSTRACT
Accelerated eutrophication in lakes reduces the number of submerged macrophytes and alters the residues of glyphosate and its degradation products. However, the effects of submerged macrophytes on the fate of glyphosate remain unclear. We investigated eight lakes with varying trophic levels along the middle and lower reaches of the Yangtze River in China, of which five lakes contained either glyphosate or aminomethylphosphate (AMPA). Glyphosate and AMPA residues were significantly positively correlated with the trophic levels of lakes (P < 0.01). In lakes, glyphosate is degraded through the AMPA and sarcosine pathways. Eight shared glyphosate-degrading enzymes and genes were observed in different lake sediments, corresponding to 44 degrading microorganisms. Glyphosate concentrations in sediments were significantly higher in lakes with lower abundances of soxA (sarcosine oxidase) and soxB (sarcosine oxidase) (P < 0.05). In the presence of submerged macrophytes, oxalic and malonic acids secreted by the roots of submerged macrophytes increased the abundance of glyphosate-degrading microorganisms containing soxA or soxB (P < 0.05). These results revealed that a decrease in the number of submerged macrophytes in eutrophic lakes may inhibit glyphosate degradation via the sarcosine pathway, leading to a decrease in glyphosate degradation and an increase in glyphosate residues.
Subject(s)
Lakes , Sarcosine , Lakes/chemistry , Sarcosine Oxidase , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid , Exudates and Transudates , China , Eutrophication , GlyphosateABSTRACT
The scandal of detecting 1, 2-propanediol (PL) in milk brought a crisis to the trust of consumers in the dairy industry, and the potential toxicity of PL has aroused the public concern about dietary exposure. A total of 200 pasteurized milk samples were collected from 15 regions, and the quantity of PL ranged between 0 and 0.31 g kg-1. Pseudo-targeted quantitative metabolomics integrated with proteomics demonstrated that PL enhanced the reduction of κ-casein, ß-casein, and 107 substances (41 amines and 66 amides) containing amide bonds. Pathway enrichment and topological analysis indicated that PL induced the metabolism of lipids, amino acids, oligosaccharide nucleotides, and alkaloids by accelerating the rate of nucleophilic reaction, and acetylcholinesterase, sarcosine oxidase, and prolyl 4-hydroxylase were determined as the vital enzymes related to the degradation of above nutrients. The results of molecular simulation calculation illustrated that the number of hydrogen bonds between acetylcholinesterase, sarcosine oxidase, and substrate increased to 2 and 3, respectively, while the position of hydrogen bonds between prolyl 4-hydroxylase and proline was shifted, indicating the change of conformation and the enhancement of hydrogen bond force were essential factors for the up-regulation of enzyme activity. This study first revealed the mechanism of deposition and transformation of PL in milk, which contributed to the knowledge of the quality control of milk and provided vital indicators to evaluate the adverse risks of PL in dairy products.
Subject(s)
Amino Acids , Propylene Glycol , Animals , Milk , Acetylcholinesterase , Sarcosine Oxidase , Amines , Propylene Glycols , Amides , Caseins , LipidsABSTRACT
An electrochemical biosensor for creatinine determination in a drop of whole human blood was developed and applied to the determination of creatinine in real clinical samples. It is based on the modification of a dual carbon working electrode with a combination of three enzymes: creatinine amidohydrolase (CNN), creatine amidinohydrolase (CRN) and sarcosine oxidase (SOX). Electrochemical transduction is performed using horseradish peroxidase (HRP) and potassium hexacyanoferrate(II) as mediator. A drop of human blood is enough to carry out the measurements by differential chronoamperometry where one carbon electrode detects creatine and the other both creatine and creatinine. The integrated differential signal obtained in the biosensor is linear with the concentration of creatinine in blood in the range 0.5-15 mg/dL and the enzyme-modified electrodes are stable for at least 3 months at 4 °C. The biosensor was lined to a reference method based on Isotope Dilution Mass Spectrometry (IDMS) with 50 real human blood samples and the results compared with those obtained by alternative routine techniques based on Jaffé method and an enzymatic method (Cobas 8000 Roche®, Crep2 Roche®). There were no significant differences between the creatinine concentrations found by the routine techniques and the developed biosensor.
Subject(s)
Biosensing Techniques , Creatine , Humans , Creatinine , Electrodes , Horseradish Peroxidase , Sarcosine Oxidase , Electrochemical TechniquesABSTRACT
Sphingomonas paucimobilis' P450SPα (CYP152B1) is a good candidate as industrial biocatalyst. This enzyme is able to use hydrogen peroxide as unique cofactor to catalyze the fatty acids conversion to α-hydroxy fatty acids, thus avoiding the use of expensive electron-donor(s) and redox partner(s). Nevertheless, the toxicity of exogenous H2 O2 toward proteins and cells often results in the failure of the reaction scale-up when it is directly added as co-substrate. In order to bypass this problem, we designed a H2 O2 self-producing enzyme by fusing the P450SPα to the monomeric sarcosine oxidase (MSOX), as H2 O2 donor system, in a unique polypeptide chain, obtaining the P450SPα -polyG-MSOX fusion protein. The purified P450SPα -polyG-MSOX protein displayed high purity (A417 /A280 = 0.6) and H2 O2 -tolerance (kdecay = 0.0021 ± 0.000055 min-1 ; ΔA417 = 0.018 ± 0.001) as well as good thermal stability (Tm : 59.3 ± 0.3°C and 63.2 ± 0.02°C for P450SPα and MSOX domains, respectively). The data show how the catalytic interplay between the two domains can be finely regulated by using 500 mM sarcosine as sacrificial substrate to generate H2 O2 . Indeed, the fusion protein resulted in a high conversion yield toward fat waste biomass-representative fatty acids, that is, lauric acid (TON = 6,800 compared to the isolated P450SPα TON = 2,307); myristic acid (TON = 6,750); and palmitic acid (TON = 1,962).
Subject(s)
Fatty Acids , Mixed Function Oxygenases , Mixed Function Oxygenases/metabolism , Sarcosine Oxidase/chemistry , Sarcosine Oxidase/metabolism , Oxidation-Reduction , Hydrogen PeroxideABSTRACT
The detection of cancer biomarkers is of great significance for the early screening of cancer. Detecting the content of sarcosine in blood or urine has been considered to provide a basis for the diagnosis of prostate cancer. However, it still lacks simple, high-precision and wide-ranging sarcosine detection methods. In this work, a Ti3 C2 TX /Pt-Pd nanocomposite with high stability and excellent electrochemical performance has been synthesized by a facile one-step alcohol reduction and then used on a glassy carbon electrode (GCE) with sarcosine oxidase (SOx ) to form a sarcosine biosensor (GCE/Ti3 C2 TX /Pt-Pd/SOx ). The prominent electrocatalytic activity and biocompatibility of Ti3 C2 TX /Pt-Pd enable the SOx to be highly active and sensitive to sarcosine. Under the optimized conditions, the prepared biosensor has a wide linear detection range to sarcosine from 1 to 1000 µM with a low limit of detection of 0.16 µM (S/N = 3) and a sensitivity of 84.1 µA/mM cm2 . Besides, the reliable response in serum samples shows its potential in the early diagnosis of prostate cancer. More importantly, the successful construction and application of the amperometric biosensor based on Ti3 C2 TX /Pt-Pd will provide a meaningful reference for detecting other cancer biomarkers.
Subject(s)
Biosensing Techniques , Prostatic Neoplasms , Humans , Male , Biomarkers, Tumor , Biosensing Techniques/methods , Carbon/chemistry , Limit of Detection , Prostatic Neoplasms/diagnosis , Sarcosine , Sarcosine Oxidase/chemistry , Titanium , Platinum , LeadABSTRACT
An unprecedented photoswitching phenomenon of flavin-inhibitor complexes in a flavoenzyme was revealed by femtosecond transient absorption spectroscopy. The vast majority of flavoenzymes, including monomeric sarcosine oxidase (MSOX), perform non-light-driven physiological functions. Yet, the participation of flavin cofactors in photoinduced electron transfer reactions is widespread. MSOX catalyzes the oxidative demethylation of sarcosine; methylthioacetate (MTA) is a substrate analog inhibitor that forms a complex with MSOX exhibiting intense absorption bands over the whole visible range due to flavin-MTA charge transfer (CT) interactions. Here, we demonstrate that upon excitation, these CT interactions vanish during a barrierless high quantum yield reaction in â¼300 fs. The initial complex subsequently geminately re-forms in a few nanoseconds near room temperature in a thermally activated way with an activation energy of 28 kJ/mol. We attribute this hitherto undocumented process to a well-defined photoinduced isomerization of MTA in the active site, as corroborated by experiments with the heavier ligand methylselenoacetate. Photoisomerization phenomena involving CT transitions may be further explored in photocatalytic and photoswitching applications of flavoenzymes.
Subject(s)
Flavins , Sarcosine , Flavins/metabolism , Kinetics , Oxidation-Reduction , Sarcosine Oxidase/chemistry , Sarcosine Oxidase/metabolismABSTRACT
In clinical practice, sera creatinine level is regarded as a crucial biomarker for the diagnosis, staging and monitoring of kidney disease. An amperometric biosensor is rapid, accurate, and cost-effective, with a portability and a simple operation. Herein, we report for the firsttime a disposable, printed amperometric biosensor for the clinical evaluation of creatinine in renal function detection. The sensor is constructed based on Prussian blue/carbon-graphite paste as the working electrode and the immobilization of creatinine amidohydrolase, creatine amidinohydrolase and sarcosine oxidase. The creatinine biosensor shows a linear detection range from 0.05 to 1.4 mM with a detection time of about 3 min. In addition, the sensor shows a high stability that can maintain above 86% of the initial activity after being stored for over 4 months. Moreover, the sensor shows almost the same results as those with the Jaffe method for measuring the real blood samples. We anticipate that the creatinine biosensor could be widely used in the medical and healthcare areas, especially for at-home testing and onsite medical examinations.
Subject(s)
Biosensing Techniques , Biosensing Techniques/methods , Creatinine , Electrodes , Enzymes, Immobilized , Kidney/physiology , Sarcosine OxidaseABSTRACT
Herein, we report a facile and label-free method for sensitive and specific determination of prostate cancer biomarker sarcosine via using photoluminescent bimetallic Au/Cu nanoplatelets (AuCu NPs) to construct an inner filter effect (IFE)-based photoluminescence (PL) sensing platform. The AuCu NPs were formed by the cysteine-induced co-reduction reaction, which displayed bright PL with an emission peak at 560 nm. Meanwhile, the Cu(I) doping caused a maximum 25-fold enhancement of quantum yield (QY), compared with the native Au(I) complexes, i.e., from 0.85 to 21.5%. By integrating the AuCu NPs with p-phenylenediamine (PPD) oxidation reaction, an IFE-based sensor for sarcosine detection was constructed. In this method, sarcosine is oxidized under the catalysis of sarcosine oxidase (SOx) to yield H2O2. The latter further oxidizes PPD to form 2,5-diamino-N,N'-bis(p-aminophenyl)-l,4-benzoquinone di-imine (PPDox) in the presence of horseradish peroxidase (HRP). The UV-vis absorption spectrum of the PPDox can overlap well with the excitation and emission spectra of the AuCu NPs, resulting in the efficient quenching of the AuCu NPs via the IFE effect. Therefore, this IFE-based AuCu NPs/SOx/PPD/HRP sensing platform can be used for highly sensitive and specific sensing of sarcosine. The sensing platform showed two linear regions of the PL intensity of the AuCu NPs versus the concentration of sarcosine in the range of 0.5-5 µM and 5-100 µM with a detection limit (LOD) of 0.12 µM (S/N = 3). Furthermore, this IFE-based sensing platform could be developed into a paper-based biosensor for simple, instrument-free, and visual detection of sarcosine.
Subject(s)
Biosensing Techniques , Sarcosine , Biomarkers, Tumor/analysis , Horseradish Peroxidase , Humans , Hydrogen Peroxide , Male , Prostatic Neoplasms , Sarcosine/analysis , Sarcosine OxidaseABSTRACT
This manuscript reports on a simple paper-based bienzymatic colorimetric assay for sarcosine as an important urinary biomarker of prostate cancer. All required assay reagents are pre-deposited on hydrophilic filter paper spots surrounded by a hydrophobic barrier. Sarcosine in the sample solution is selectively oxidized in the presence of sarcosine oxidase (SOx), resulting in the formation of hydrogen peroxide, which is subsequently detected through the horseradish peroxidase (HRP)-catalyzed conversion of the colorless indicator 3,3',5,5'-tetramethylbenzidine (TMB) into its blue-colored oxidation product. By the modification of the paper with positively charged poly(allylamine hydrochloride) (PAH), a linear response to sarcosine between 0 and 10 µM and a significant lowering of the limit of detection (LOD) (0.6 µM) compared to the unmodified paper substrate (12.6 µM) has been achieved. The improvement of the LOD was attributed to the fact that the presence of the polymer limits the enzyme-driven colorimetric reaction to the surface of the paper substrate, resulting in stronger color development. In experiments in artificial urine matrix, the bicarbonate anion was identified as an inhibitor of the colorimetric reaction. This inhibition was successfully eliminated through on-device sample pH adjustments with pH-buffer components pre-deposited onto assay devices. The LOD for sarcosine achieved in artificial urine matrix (2.5 µM) is below the 5 µM threshold value for this urinary biomarker required for diagnostic purposes. Finally, good selectivity over all 20 natural amino acids and satisfactory long-term storage stability of reagent-modified paper substrates at - 20 °C for a period of 50 days were confirmed.
Subject(s)
Colorimetry , Sarcosine , Colorimetry/methods , Horseradish Peroxidase , Humans , Hydrogen Peroxide , Limit of Detection , Male , Sarcosine Oxidase/chemistryABSTRACT
Rapid and specific identification of tumor metabolic markers is of great significance. Herein, a convenient, reliable and specific strategy was proposed to screen prostate cancer (PCa) individuals through indirectly quantifying sarcosine, an early indicator of PCa, in the clinical urine samples. The success roots in the rational design of a cascade response model, which takes integrated sarcosine oxidase (SOX) as a specific recognition unit and oxygen-sensitive molecule as a signal reporter. The newly developed hierarchical mesoporous Zr-based metal-organic frameworks with continuously tunable mesopore size ensure the synergetic work of the SOX and response unit spatially separated in their neighboring mesoporous and microporous domains, respectively. The large mesopore up to 12.1 nm not only greatly enhances the loading capacity of SOX but also spares enough space for the free diffusion of sarcosine. On this basis, the probe is competent to specifically check out the tiny concentration change of sarcosine in the urine sample between PCa patients and healthy humans. Such a concept of enzyme-assisted substrate sensing could be simply extended by altering the type of immobilized enzymes, hopefully setting a guideline for the rational design of multiple probes to quantify specific biomarkers in complex biological samples.
Subject(s)
Electrochemical Techniques/methods , Metal-Organic Frameworks/chemical synthesis , Prostatic Neoplasms/diagnosis , Biomarkers, Tumor , Humans , Limit of Detection , Male , Metal-Organic Frameworks/chemistry , Models, Molecular , Molecular Structure , Sarcosine Oxidase/chemistry , Sarcosine Oxidase/metabolismABSTRACT
BACKGROUND: Serum creatinine is a widely used biomarker for evaluating renal function. Sarcosine oxidase enzymatic (SOE) analysis is currently the most widely used method for the detection of creatinine. This method was negatively interfered with by calcium dobesilate, causing pseudo-reduced results. The aim of this study was to explore a new method to alleviate the negative interference of this drug on creatinine detection. METHOD: We formulated eight drug concentrations and 12 creatinine concentrations from serum. The SOE method, the new method, and the Jaffe method were used for detection in five systems. Creatinine biases were analyzed under the conditions with or without the interference of calcium dobesilate, at consistent or inconsistent creatinine concentrations. Creatinine concentrations were also analyzed at three medical decision levels (MDLs). RESULTS: Calcium dobesilate had negative interference in creatinine SOE analysis. With the increase in calcium dobesilate concentrations, the negative bias increases. The new BG method showed an anti-negative interference effect. In the Roche system, the BG method reduced the negative bias from -71.11% to -16.7%. In the Abbott system, bias was reduced from -45.15% to -2.74%. In the Beckman system, the bias was reduced from -65.36% to -7.58%. In the Siemens system, the bias was reduced from -58.62% to -7.58%. In the Mindray system, the bias was reduced from -36.29% to -6.84%. CONCLUSION: The new method alleviated the negative interference of calcium dobesilate in creatinine SOE detection. The negative bias could be reduced from -60% or -70% to less than -20%.
Subject(s)
Biomarkers/blood , Calcium Dobesilate/pharmacology , Clinical Enzyme Tests/methods , Creatinine/blood , Kidney Diseases/diagnosis , Sarcosine Oxidase/drug effects , Artifacts , Blood Chemical Analysis , Hemostatics/pharmacology , Humans , Kidney Diseases/blood , Kidney Function Tests , Sarcosine Oxidase/bloodABSTRACT
A nano-integrated portable enzymatic microfluidic electrochemical biochip was developed for single-step point-of-care testing of creatinine. The biochip could automatically eliminate a lot of interferences from practical biological samples and enzymatic intermediate products. Gold nanostructure- and carbon nanotube-based screen-printed carbon electrodes were integrated into microfluidic structures to improve the detection performance for creatinine. The microfluidic electrochemical biochip holds promise to become a practical device for medical diagnosis, especially POCT.
Subject(s)
Creatinine/blood , Electrochemical Techniques , Lab-On-A-Chip Devices , Nanotechnology , Point-of-Care Systems , Horseradish Peroxidase/metabolism , Humans , Particle Size , Sarcosine Oxidase/metabolism , Ureohydrolases/metabolismABSTRACT
The subfamily of sarcosine oxidase is a set of enzymes within the larger family of amine oxidases. It is ubiquitously distributed among different kingdoms of life. The member enzymes catalyze the oxidization of an N-methyl amine bond of amino acids to yield unstable imine species that undergo subsequent spontaneous non-enzymatic reactions, forming an array of different products. These products range from demethylated simple species to complex alkaloids. The enzymes belonging to the sarcosine oxidase family, namely, monomeric and heterotetrameric sarcosine oxidase, l-pipecolate oxidase, N-methyltryptophan oxidase, NikD, l-proline dehydrogenase, FsqB, fructosamine oxidase and saccharopine oxidase have unique features differentiating them from other amine oxidases. This review highlights the key attributes of the sarcosine oxidase family enzymes, in terms of their substrate binding motif, type of oxidation reaction mediated and FAD regeneration, to define the boundaries of this group and demarcate these enzymes from other amine oxidase families.