Distance-based analytical device integrated with carbon nanomaterials for sarcosine quantification in human samples.

A straightforward distance-based paper analytical device (dPAD) was developed for monitoring sarcosine levels in human samples for the rapid diagnosis and prognosis of prostate cancer and related symptoms. This assay eliminates the need for the expensive horseradish peroxidase (HRP) enzyme by utilizing carbon nanodots (CDs) as a peroxidase-like nanozyme. The proposed dPAD sensor consists of a sample zone pre-deposited with sarcosine oxidase (SOx) and CDs, and a detection zone containing 3,3',5,5'-tetramethylbenzidine (TMB). When a solution containing sarcosine is added to the sample zone, hydroxyl radicals (•OH) are produced through SOx oxidation and subsequent peroxidase catalysis by the CDs. The formed •OH radicals immediately flow to the detection zone via capillary force, where they oxidize TMB, resulting in a visible colour change from colourless to blue. Sarcosine quantification is effortlessly accomplished by measuring the distance of the blue colour in the detection zone. The developed dPAD offers a linear working range between 12.5 and 35.0 nmol L-1 (R2 = 0.9959) and a detection limit (LOD) of 10.0 nmol L-1. This covers the clinical range for urinary sarcosine determination, thereby suggesting no additional sample preparation or dilution is needed. The sensor shows high precision with the highest relative standard deviation (RSD) of 4.58% and demonstrates excellent selectivity with no observed interferences. Furthermore, recovery studies in human control urine samples ranged from 98.67 to 101.50%, with the highest RSD of 2.03%. Correspondingly, our dPAD method showed a great match with the performance of a commercial ELISA method for detecting sarcosine in human control serum. The sensor is more cost-effective, user-friendly, and accessible than previous methods. Overall, the proposed method represents a promising analytical tool for sarcosine quantification. The concept is also applicable for broader analytical applications in detecting other biomolecules.

Point-of-care testing device platform for the determination of creatinine on an enzyme@CS/PB/MXene@AuNP-based screen-printed carbon electrode.

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).

Natural Protein Photon Upconversion Supramolecular Assemblies for Background-Free Biosensing.

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 (∼8.2 ± 1.1 nm), which were further assembled into photon upconversion supramolecular assemblies (PUSA). This PUSA exhibited unique features, including a small size (∼44.1 ± 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.

Dual-mode fluorescence and colorimetric smartphone-based sensing platform with oxidation-induced self-assembled nanoflowers for sarcosine detection.

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.

CYP116B5-SOX: An artificial peroxygenase for drug metabolites production and bioremediation.

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.

Chemical modification of Arthrobacter sarcosine oxidase by N-methylisothiazolinone reduces reactivity toward oxygen.

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.

Construction of Double-enzyme Complexes with DNA Framework Nanorulers for Improving Enzyme Cascade Catalytic Efficiency.

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 5.67 nm to 12.33 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.

Design of a H2 O2 -generating P450SPα fusion protein for high yield fatty acid conversion.

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).

A Ti3 C2 TX /Pt-Pd based amperometric biosensor for sensitive cancer biomarker detection.

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.

Photoswitching Behavior of Flavin-Inhibitor Complex in a Nonphotocatalytic Flavoenzyme.

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 ∼300 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.

Colorimetric paper-based sarcosine assay with improved sensitivity.

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.

Specific Screening of Prostate Cancer Individuals Using an Enzyme-Assisted Substrate Sensing Platform Based on Hierarchical MOFs with Tunable Mesopore Size.

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.

The family of sarcosine oxidases: Same reaction, different products.

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.

Potential of mean force and umbrella sampling simulation for the transport of 5-oxazolidinone in heterotetrameric sarcosine oxidase.

The structure of heterotetrameric sarcosine oxidase (HSO) contains a highly complex system composed of a large cavity and tunnels, which are essential for the reaction and migration of the reactants, products, and intermediates. Previous geometrical analysis using the CAVER program has predicted that there are three possible tunnels, T1, T2, and T3, for the exit pathway of the iminium intermediate, 5-oxazolidinone (5-OXA), of the enzyme reaction. Previous molecular dynamics (MD) simulation of HSO has identified the regions containing the water channels from the density distribution of water. The simulation indicated that tunnel T3 is the most probable exit pathway of 5-OXA. In the present study, the potential of mean force (PMF) for the transport of 5-OXA through tunnels T1, T2, and T3 was calculated using umbrella sampling (US) MD simulations and the weighted histogram analysis method. The PMF profiles for the three tunnels support the notion that tunnel T3 is the exit pathway of 5-OXA, and that 5-OXA tends to stay at the middle of the tunnel. The maximum errors of the calculated PMF for the predicted exit pathway, tunnel T3, were estimated by repeating the US simulations using different sets of initial positions. The PMF profile was also calculated for the transport of glycine within T3. The PMF profiles from the US simulations were in good agreement with the previous predictions that 5-OXA escape through tunnel T3 and how glycine is released to the outside of HSO was discussed.

Iron doped graphitic carbon nitride with peroxidase like activity for colorimetric detection of sarcosine and hydrogen peroxide.

The successful synthesis is reported of Mn, Fe, Co, Ni, Cu-doped g-C3N4 nanoflakes via a simple one-step pyrolysis method, respectively. Among them, the Fe-doped g-C3N4 nanoflakes exhibited the highest peroxidase-like activity, which can be used for colorimetric detection of hydrogen peroxide (H2O2) and sarcosine (SA), within the detection ranges of 2-100 µM and 10-500 µM and detection limits of 1.8 µM and 8.6 µM, respectively. The catalytic mechanism of the Fe-doped g-C3N4 nanoflake was also explored and verified the generation of hydroxyl radical (•OH) through fluorescence method. It is believed that the Fe-doped g-C3N4 nanoflakes as enzyme mimics will greatly promote the practical applications in a variety of fields in the future including biomedical science, environmental governance, antibacterial agent, and bioimaging due to their extraordinary catalytic performance and stability. Graphical abstract.

Facile one-step targeted immobilization of an enzyme based on silane emulsion self-assembled molecularly imprinted polymers for visual sensors.

Immobilized enzymes play significant roles in many practical applications. However, the enzymes need to be purified before immobilization by conventional immobilizing methods, and the purification process is expensive, laborious, complicated and results in a decrease of the enzymatic activity. So, we present a novel method by a facile one-step targeted immobilization of an enzyme without a purification process from complex samples. For this purpose, a novel molecularly imprinted polymer was prepared via a silane emulsion self-assembly method using boric acid-modified Fe3O4 nanoparticles as magnetic nuclei, horseradish peroxidase as a template, 3-aminopropyltriethoxysilane as a functional monomer and tetraethyl orthosilicate as a crosslinking agent. The molecularly imprinted polymers were characterized using a scanning electron microscope, X-ray photoelectron spectroscope, vibrating sample magnetometer and X-ray diffractometer. The as-prepared and characterized materials were employed to immobilize horseradish peroxidase from a crude extract of horseradish. Moreover, the immobilized horseradish peroxidase was employed to develop visual sensors for the detection of glucose and sarcosine. This study demonstrated that the molecularly imprinted polymers prepared via the silane emulsion self-assembly method can facilely immobilize horseradish peroxidase from a crude extract of horseradish without any purification process. The developed visual method based on the immobilized horseradish peroxidase shows great potential applications for the visual detection of glucose and sarcosine.

Amperometric sarcosine biosensor based on hollow magnetic Pt-Fe3O4@C nanospheres.

Sarcosine is a recently identified biomarker for prostate cancer. However, the rapid detection methods for sarcosine are relatively lack because of the low concentration and the presence of complicated interfering substances in serum or urine. In this manuscript, hollow nanospheres of Fe3O4 was synthesized and used as carrier to disperse Pt (Pt) nanoparticles. In order to achieve excellent electron transfer ability, we use polyaniline to coat Pt-Fe3O4 nanoparticles, and pyrolyze the polyaniline to carbon (C). Thus, hollow magnetic Pt-Fe3O4@C nanocomposites with good electron transfer ability are formed. The Pt-Fe3O4@C nanocomposites have high catalytic activity and stability. The nanocomposites were immobilized on glassy carbon electrode (GCE) to construct a nonenzyme hydrogen peroxide (H2O2) sensor (Pt-Fe3O4@C/GCE). We further construct a sensitive sarcosine biosensor by immobilizing sarcosine oxidase (SOx) on the Pt-Fe3O4@C/GCE. The high catalytic activity and good biocompatibility of Pt-Fe3O4@C nanocomposites greatly retained the bioactivity of immobilized SOx, and the prepared sarcosine biosensor has good electrocatalytic performance towards sarcosine. It has a linear detection range between 0.5 and 60 µM with a limit of detection (LOD) of 0.43 µM (the signal to noise ratio is 3), and the sensitivity is 3.45 nA µM-1 (48.8 nA µM-1 cm-2), which has the potential to be used for rapid screening of prostate cancer.

Multiplexed Readout of Enzymatic Reactions by Means of Laterally Resolved Illumination of Quantum Dot Electrodes.

Triggering electrochemical reactions with light provides a powerful tool for the control of complex reaction schemes on photoactive electrodes. Here, we report on the light-directed, multiplexed detection of enzymatic substrates using a nonstructured gold electrode modified with CdSe/ZnS quantum dots (QDs) and two enzymes, glucose oxidase (GOx) and sarcosine oxidase (SOx). While QDs introduce visible-light sensitivity into the electrode architecture, GOx and SOx allow for a selective conversion of glucose and sarcosine, respectively. For the QD immobilization to the gold electrode, a linker-assisted approach using trans-4,4'-stilbenedithiol has been used, resulting in the generation of a photocurrent. Subsequently, GOx and SOx have been immobilized in spatially separated spots onto the QD electrode. For the local readout of the QD electrode, a new measurement setup has been developed by moving a laser pointer across the surface to defined positions on the chip surface. The amplitudes of the photocurrents upon illumination of the GOx or SOx spot depend in a concentration-dependent manner on the presence of glucose and sarcosine, respectively. This measurement also allows for a selective detection in the presence of other substances. The setup demonstrates the feasibility of multiplexed measurements of enzymatic reactions using a focused light pointer, resulting in an illumination area with a diameter of 0.3 mm for analyzing spots of different enzymes. Moving the laser pointer in the x- and y-direction and simultaneously detecting the local photocurrent also allow a spatial imaging of enzyme immobilization. Here, not only the spot dimensions but also the activity of the enzyme can be verified.

A Rapid Method for the Detection of Sarcosine Using SPIONs/Au/CS/SOX/NPs for Prostate Cancer Sensing.

BACKGROUND: Sarcosine is an amino acid that is formed by methylation of glycine and is present in trace amounts in the body. Increased sarcosine concentrations in blood plasma and urine are manifested in sarcosinemia and in some other diseases such as prostate cancer. For this purpose, sarcosine detection using the nanomedicine approach was proposed. In this study, we have prepared superparamagnetic iron oxide nanoparticles (SPIONs) with different modified surface area. Nanoparticles (NPs) were modified by chitosan (CS), and sarcosine oxidase (SOX). SPIONs without any modification were taken as controls. Methods and Results: The obtained NPs were characterized by physicochemical methods. The size of the NPs determined by the dynamic light scattering method was as follows: SPIONs/Au/NPs (100⁻300 nm), SPIONs/Au/CS/NPs (300⁻700 nm), and SPIONs/Au/CS/SOX/NPs (600⁻1500 nm). The amount of CS deposited on the NP surface was found to be 48 mg/mL for SPIONs/Au/CS/NPs and 39 mg/mL for SPIONs/Au/CS/SOX/NPs, and repeatability varied around 10%. Pseudo-peroxidase activity of NPs was verified using sarcosine, horseradish peroxidase (HRP) and 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. For TMB, all NPs tested evinced substantial pseudo-peroxidase activity at 650 nm. The concentration of SPIONs/Au/CS/SOX/NPs in the reaction mixture was optimized to 0⁻40 mg/mL. Trinder reaction for sarcosine detection was set up at 510 nm at an optimal reaction temperature of 37 °C and pH 8.0. The course of the reaction was linear for 150 min. The smallest amount of NPs that was able to detect sarcosine was 0.2 mg/well (200 µL of total volume) with the linear dependence y = 0.0011x - 0.0001 and the correlation coefficient r = 0.9992, relative standard deviation (RSD) 6.35%, limit of detection (LOD) 5 µM. The suggested method was further validated for artificial urine analysis (r = 0.99, RSD 21.35%, LOD 18 µM). The calculation between the detected and applied concentrations showed a high correlation coefficient (r = 0.99). NPs were tested for toxicity and no significant growth inhibition was observed in any model system (S. cerevisiae, S. aureus, E. coli). The hemolytic activity of the prepared NPs was similar to that of the phosphate buffered saline (PBS) control. The reaction system was further tested on real urine specimens. Conclusion: The proposed detection system allows the analysis of sarcosine at micromolar concentrations and to monitor changes in its levels as a potential prostate cancer marker. The whole system is suitable for low-cost miniaturization and point-of-care testing technology and diagnostic systems. This system is simple, inexpensive, and convenient for screening tests and telemedicine applications.

Construction and application of amperometric sarcosine biosensor based on SOxNPs/AuE for determination of prostate cancer.

An improved amperometric sarcosine biosensor was constructed based on covalent immobilization of sarcosine oxidase nanoparticles (SOxNPs) onto gold electrode (AuE). The SOxNPs/AuE was characterized by scanning electron microscopy (SEM), fourier transform infrared (FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS) at different stages of its construction. The biosensor worked optimally within 2 s at a potential of 1.0 V, against Ag/AgCl, pH 6.5 and 35 °C. A linear relationship was observed between sarcosine concentration range, 0.1-100 µM and the biosensor response i.e. current in mA under optimum conditions. The biosensor offered a low detection limit of 0.01 µM and gratifying storage stability. The SOxNPs/AuE was unaffected by a number of serum substances at their physiological concentrations. The biosensor measured sarcosine level in sera collected from persons suffering from prostate cancer (mean13.5 µM, n = 8), which was significantly higher (p < 0.01) than those in apparently healthy persons (mean 2.2 µM, n = 8). The SOxNPs/Au electrode was reused 300- times during the span of 180 days, with only 10% loss in its initial activity while being stored dry at 4 °C.