Detection Of Homocysteine Research Articles

A benzotriazole-coumarin derivative as a turn-on fluorescent probe for highly efficient and selective detection of homocysteine and its bioimaging application

Biological thiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play essential roles in physiological and pathological processes, abnormal concentration of biothiols could lead to various diseases. Due to the structure similarity, it’s difficult to design fluorescence probes to selectively detect the common biothiols. Herein, a series of fluorescent molecules based on benzotriazole and coumarin moieties were designed and synthesized. Among these molecules, (E)-3-(2-(1H-benzo[d][1–3]triazol-1-yl)vinyl)-4-chloro-7-(diethylamino)–2H-chromen-2-one (BTAC (e)) exhibits high selectivity towards Hcy. When different biothiols were introduced, the probe BTAC(e) could react with Hcy by a thiol-halogen SNAr nucleophilic substitution-rearrangement reaction and exhibited enhancement in fluorescence. With the introduction of benzotriazole, BTAC(e) could respond to Hcy more quickly. At the same time, this probe exhibited excellent selectivity towards Hcy, it turns out that Cys and GSH have little influence on the fluorescence of BTAC(e). Under the optimized conditions, the fluorescence intensity has a good linear relationship with the concentration of Hcy in the range of 2.0 × 10-7 to 7.0 × 10-6 mol L-1 and the limit of detection for Hcy is 1.55 × 10-8 mol L-1. Moreover, probe BTAC (e) has been applied for imaging Hcy in living cells and the experimental result is satisfactory.

Folic acid incorporated nitrogen-doped carbon dots as a turn-on fluorescence probe for homocysteine detection.

This study describes the development of a low-cost fluorescence assay for detecting homocysteine (Hcy) without the interference of cysteine and glutathione using carbon quantum dots. Herein nitrogen-doped carbon quantum dots (NCDs) were synthesized from citric acid as the carbon source and urea as the dopant using a one-pot microwave-assisted method. The obtained NCDs were incorporated with folic acid (FA) by the direct ex situ addition method and were used as a fluorescence probe to detect Hcy. The probe exhibited a fluorescence turn-on response with increased Hcy concentration up to 50 μM with a limit of detection of 2.276 μM. The point of care detection of Hcy using the probe was also tested with a paper-based assay strip.

Synthesis and Characterization of 1H-Imidazole-4,5-dicarboxylic Acid-Functionalized Silver Nanoparticles: Dual Colorimetric Sensors of Zn2+ and Homocysteine.

A colorimetric assay has been developed for Zn2+ and homocysteine (Hcy) detection using functionalized silver nanoparticles (AgNPs). AgNPs have been synthesized using silver nitrate, where sodium citrate is used as a stabilizing agent and NaBH4 as a reducing agent. Then, the nanoparticles (citrate@AgNPs) were functionalized with 1H-imidazole-4,5-dicarboxylic acid (IDCA). UV–visible and FTIR spectra suggested that IDCA was functionalized on the surface of citrate@AgNPs through the N atom of the imidazole ring. The IDCA-functionalized silver nanoparticles (IDCA@AgNPs) simultaneously detected Zn2+ and Hcy from aqueous solution and showed different responses to the two analytes (Zn2+ and Hcy) based on the aggregation-induced color change of IDCA@AgNPs. They showed the color change from yellow to red, which was easily discriminated by visual inspection as well as UV–visible spectroscopy. The surface plasmon resonance absorbance values of Zn2+ and Hcy are 485 and 512 nm, respectively, when Zn2+ and Hcy react with IDCA@AgNPs. IDCA@AgNPs showed linearity with Zn2+ and Hcy concentrations, with the detection limit of 2.38 μM and 0.54 nM, respectively (S/N = 3). The IDCA@AgNPs showed excellent selectivity toward Zn2+ and Hcy compared to the different metal ions and amino acids, respectively. Optimal detection was achieved toward Zn2+ and Hcy in the pH range 3–10. In addition, IDCA@AgNPs were used to detect Zn2+ and Hcy from lake water, showing low interference.

Colorimetric determination of biothiols based on peroxidase-mimicking Ag nanoparticles decorated Ti3C2 nanosheets.

Ag nanoparticle-decorated Ti3C2 nanosheets (AgNPs@Ti3C2 NSs) were facilely synthesized via a self-reduction approach, in which Ti3C2 NSs acted as both reductant and supporter. The AgNPs@Ti3C2 NS nanocomposite exhibited excellent peroxidase-like activity with o-phenylenediamine (OPD) and H2O2 as substrates. The catalytic behavior followed the typical Michaelis-Menten kinetics; Michaelis constant (Km) and maximum initial velocity (Vmax) for OPD were 0.263mM and 43.2 × 10-8M-1s, indicating high affinity and high catalytic efficiency towards OPD. The catalytic mechanism was revealed to be an accelerated electron transfer process. Based on the inhibition effect on the peroxidase-like activity of AgNPs@Ti3C2 NSs, a simple, fast, and sensitive colorimetric method for detection of low-weight biothiols (cysteine (Cys), homocysteine (Hcy), and glutathione (GSH)) was developed by measuring the absorbance at 425nm. The colorimetric method displayed wide linear range (50nM to 50μM for Cys, 10nM to 250μM for Hcy, 10nM to 50μM for GSH), low limit of detection (48.5nM for Cys, 5.5nM for Hcy, 7.0nM for GSH), and good selectivity and short assay time (3min). Moreover, the feasibility of this colorimetric sensor was demonstrated by accurately determining Cys in diluted human serum samples; good recovery (95.9-101.0%) and low relative standard deviations (2.8-4.9%) were obtained, showing great promise for point-of-care test in clinical samples.

Colorimetric and photoluminescent probes based on iridium(III) complexes for highly selective detection of homocysteine

As a biological thiol closely related to the incidence of gastric cancer, homocysteine (Hcy) has been reported to be detected in vivo and in vitro. The design of ratiometric photoluminescent probes often relies on the unique chemical structure of the molecular. In this paper, we designed and synthesized three aldehyde-containing iridium(III) complexes and characterized their structure and photophysical properties. They showed a good specificity for Hcy detection. The color of the solution gradually changed from red to pale yellow. The photoluminescent variation from red to green was observed by the naked eye after addition of Hcy to the solution of the Ir1 ([(CHO-pba)2Ir(bpy-2NH2)]PF6), Ir2 ([(CF3-pba)2Ir(bpy-2NH2)]PF6) and Ir3 ([(CH3-pba)2Ir(bpy-2NH2)]PF6) with a large blue shift by 120, 63 and 106 nm, respectively. Based on the differences in their structures, it could be inferred that this type of ratiometric photoluminescent probe based on iridium(III) complexes with aldehyde was related to the strong electron-donating effect of the nitrogen-containing substituents on the bipyridine ligand in the structure, and the introduction of electron-donating groups on the 4-(2-pyridinyl)benzaldehyde (pba) ligand can enlarge the blue shift of the photoluminescent wavelength upon sensing of Hcy. On the other hand, iridium(III) complexes are currently recognized as a third-generation good photodynamic therapy photosensitizer. The photodynamic therapy properties of the three iridium(III) complexes in this article have also been evaluated. All of them have strong singlet oxygen generation ability and low cytotoxicity which are expected to be used in photodynamic therapy and Hcy detection.

Probing Biothiols Using a Red-Emitting Pyridoxal Derivative by Adopting Copper(II) Displacement Approach and Cell Imaging.

An aggregation-induced emission (AIE) active Schiff base L was obtained by reacting pyridoxal and 2-hydroxy-1-naphthaldehyde with p-phenylenediamine in two simple steps. The colorimetric, UV/VIS and fluorescence studies of L revealed that the yellow emissive L (λem =540 nm, λex =450 nm) in pure DMSO turned to a red-emissive L, when the poor solvent fraction (HEPES buffer, 10 mM, pH 7.4) was increased above 50 % in DMSO. The SEM and DLS results indicated the formation of self-aggregates of L that restricted the intramolecular motion and promoted the excited state intramolecular proton transfer (ESIPT) process. The cations sensing ability of the AIEgen L was explored in HEPES buffer (5 % DMSO, 10 mM, pH 7.4), where Cu2+ selectively quenched the fluorescence at 608 nm due to the chelation-enhanced fluorescence quenching (CHEQ) effect with an estimated sensitivity limit of 0.9 μM. Subsequently, the in situ formed AIEgen L-Cu2+ complex was applied for the cascade detection of glutathione (GSH), cysteine (Cys) and homocysteine (Hcy). The decomplexation of Cu2+ from the AIEgen L-Cu2+ by GSH, Cys and Hcy restored the quenched fluorescence emission of AIEgen L at 608 nm. With this Cu2+ displacement approach, the concentration of Cys, Hcy and GSH can be detected down to 2.8 μM, 3.12 μM and 2.0 μM, respectively. The practical utility of AIEgen L and AIEgen L-Cu2+ was examined by monitoring the selective analytes in real environmental and biological samples, and also applied successfully for the cell imaging applications.

A Label-Free Molecularly Imprinted Electrochemical Sensor Based on MXene Nanosheets Modified by Gold Nanoparticles for Sensitive and Selective Detection of Homocysteine

A label-free molecularly imprinted electrochemical sensor (MIECS) based on electropolymerized molecularly imprinted polymer (MIP) was developed for the determination of homocysteine (Hcy) in serum for the first time. MXene@AuNPs with layered structure was synthesized on the electrode by modifying MXene with gold nanoparticles (AuNPs). MIP based on dopamine hydrochloride (DA) were electropolymerized onto the surface of MXene@AuNPs modified electrode by molecular docking and quantum chemical calculations for specific recognition of Hcy. MXene@AuNPs as a carrier for immobilizing MIP steadily enhances the conductivity of the electrode (about 4.2-fold) and plays a crucial part in improving the detection sensitivity of MIECS. The results showed that the current response linearly decreased with the increasing concentration of Hcy in the detection range from 1 × 10−13 to 1 × 10−5 mol l−1, the limit of detection (LOD) and limit of quantification (LOQ) of 11.81 fmol l−1 and 39.49 fmol l−1, respectively. With favorable selectivity, stability, reproducibility and ruggedness, the developed MIECS was applied to the determination of Hcy in human serum samples with recoveries of 87.83%–92.58%. The proposed strategy has potential application for disease surveillance.

Risk Factor Analysis of Hepatic Encephalopathy and the Establishment of Diagnostic Model.

To identify laboratory diagnostic indicators of hepatic encephalopathy (HE), the present study established a HE diagnostic model to explore the diagnostic value of serum homocysteine, lactic acid, procalcitonin, and bile acid levels in HE identification. 371 patients with liver cirrhosis were selected as research objects, who were admitted to the Department of Hepatic Diseases, Affiliated Hospital of Northwest Minzu University from August 2019 to August 2020. The Spearman correlation results indicated that between lactic acid, procalcitonin, bile acid, serum homocysteine, and HE, the coefficients were -0.15, 0.41, 0.29, and -0.19, respectively. Univariate and multivariate analysis methods were adopted for inpatient analysis to identify the influencing factors of HE occurrence, and the diagnosis of the HE identification model was subsequently constructed. The univariate logistic regression showed that risk of developing HE increased as bile acid level (P = 0.00434) and serum homocysteine (P = 0.058) increased. Multivariate logistic regression diagnostic model of bile acid level and serum homocysteine revealed that the AUC value of the area under the ROC curve was 0.7201, indicating that the diagnostic model produced a satisfactory evaluation effect. The model formula referred logistic (P) = −2.4544 + 0.0117 bile acid levels + 0.0198 serum homocysteine. In this study, the HE diagnostic model was established using logistic regression analysis, which could benefit patients in early HE differential diagnosis. Particularly, combined detection of serum homocysteine and bile acid levels was considered to be more significant.

Application Value of Serum Hcy, TLR4, and CRP in the Diagnosis of Cerebral Small Vessel Disease.

Objective To evaluate the application value of combined detection of serum homocysteine (Hcy), Toll-like receptor 4 (TLR4), and C-reactive protein (CRP) in the diagnosis of cerebral small vessel disease (CSVD). Methods 90 patients with CSVD admitted to our hospital within the past year were identified as the research subjects, and the patients with cognitive dysfunction were assigned to the experimental group, and those with normal cognitive function were assigned to the control group according to the evaluation of cognitive dysfunction by the Montreal Cognitive Assessment (MoCA), with 45 cases in each group. Results The experimental group obtained remarkably elevated Hcy levels than the control group (P < 0.05). The patient's cognitive dysfunction is mainly attributed to the impact of serum Hcy. TLR4 and Hcy were negatively correlated with MoCA scores (P > 0.05). In comparison with the control group, the experimental group had significantly higher levels of Hcy, serum CRP, and interleukin (IL)-6 (P < 0.05). Conclusion The combined detection of serum Hcy, TLR4, and CRP features a high clinical value in the diagnosis of CSVD, which contributes to the prevention and treatment of cognitive dysfunction in patients.

Time-dependent NIR fluorescent probe with large Stokes-shift for detecting Cys/Hcy and cell imaging

Detection and imaging of Cysteine (Cys) and Homocysteine (Hcy) in living systems have drawn extensive attention over the years due to their significant biological role. However, few fluorescence probes have been reported to distinguish Cys and Hcy in near-infrared (NIR) range. In this paper, an original NIR Cys and Hcy fluorescent probe, namely DTRN, was synthesized. DTRN was constructed by using an acryloyl ester group as a trigger and dicyanoisophorone-derived as the fluorophore unit, it was found to emit NIR fluorescence with a 718 nm emission and have large stokes shift (∼176 nm). DTRN hold excellent performances such as ultrasensitivity (LOD = 0.09 μM), high specificity and fast response (<2 min) for Cys. Distinctly, after Hcy was introduced, the emission intensity of DTRN at 718 nm must take 20 min to reach a plateau and the LOD was found to be 0.12 μM. The sensing mechanism for Cys and Hcy in C2H5OH/PBS (4/6, v/v, pH = 7.4) was studied by 1H NMR spectroscopy and HRMS. Most importantly, the ability of DTRN to detect Cys in HeLa cells had also been achieved.

Detection of glutathione, cysteine, and homocysteine by online derivatization-based electrospray mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) is one of the most popular techniques for obtaining structural information, which is commonly used in bioanalysis and clinical diagnostics. However, for the detection of complicated samples with high reactivities (such as reactive sulfur species, RSS), traditional ESI-MS usually suffers from overlapped and inaccurate signals. In this study, based on the multiphase flow of extractive electrospray ionization (MF-EESI), an ambient MS technique of online derivatization was proposed to detect thiols without any other sample pretreatment. RSS molecules and the derivatization reagent of 4-chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl) were introduced into the internal and innermost capillary of the MF-EESI system, respectively. By a high-velocity nebulizing stream of N2 gas through an external capillary, both flows of innermost biothiols and internal NBD-Cl were electrosprayed and mixed for online reactions. Therefore, the fast derivatization of thiols was used to generate stable ionized derivatives for MS detection. By evaluating the changes in MS signals before and after the derivatization, the ions of RSS were identified simply and correctly. Without any sample pretreatment, the fast detection of cysteine, homocysteine, and glutathione has been achieved in the complicated samples. The present online derivatization-based MF-EESI was successfully used for fast, simple, and accurate detection of biothiols. This presented a potential pathway for the fast identification of thiols in complicated samples.

Fluorescent determination of cysteine and homocysteine via adjustable synthesis of flower-shaped covalent organic frameworks

Detection of cysteine (Cys) and homocysteine (Hcy) is very important for the early diagnosis and prevention of related diseases. Herein, we propose a facile, green, room-temperature controllable approach for the synthesis of fluorescent covalent organic frameworks (COFs) with high specific surface areas, high crystallinity, good morphology, excellent chemical and thermal stability by using 2,5-dimethoxyterephthaldehyde and 1,3,5-tris(4-aminophenyl)benzene as precursors. Interestingly, we found that the size and morphology of COFs could be controlled by the amount of acetic acid. With increasing numbers of acetic acid (HAc) molecules, the morphology of COFs gradually changed from spherical to flower-shaped, and the particle size gradually decreased from micrometer to nanometer. Furthermore, we were surprised to find that the reaction time influenced the uniform dispersion and crystallinity of flower-shaped COFs. With increasing the reaction time, the boundary around COFs became blurred, hierarchical flower-shaped COFs gradually appeared, and the flower-shaped gradually became clear and uniform. On this basis, the flower-shaped COFs were further used for highly sensitive determination of Cys and Hcy, with detection limits as low as 11.4 nM and 6.7 nM, respectively. The possible quenching mechanism was deduced by the Dmol3 package provided by Materials Studio and DFT calculations of Gaussian 09 software. This strategy could provide us a new insight into the controllable preparation of flower-shaped fluorescent COFs and broaden the application range of COFs for biomarker detection.

Simple and fast detection of homocysteine by cucurbit[7]uril fluorescent probe based on competitive strategy

This research put forward a simple and rapid fluorescence probe method based on competition strategy to detect homocysteine. The host–guest interaction behavior between cucurbit[7]uril and homocysteine was further studied via UV–vis spectroscopy, Fourier transform infrared spectroscopy, molecular modeling, and 1H NMR spectroscopy in aqueous solution. Under the optimal detection conditions, the fluorescence intensity of the cucurbit[7]uril-berberine complex declined linearly with increasing concentration of homocysteine in the range from 5 to 200 µM, and the limit of detection was 1.76 µM. Meanwhile, it was also successfully employed to detect homocysteine in serum samples with the recovery rate of 85.1% to 102.0%.

Development of a colloidal gold strip assay for the detection of total homocysteine in serum samples.

Homocysteine (Hcy) is an amino acid found in the human body and has the potential to predict cardiovascular risk where timely detection and treatment are very important. In this study, we established a method to combine enzymatic hydrolysis with a colloidal gold lateral flow immunoassay (LFIA) for the detection of total Hcy (tHcy) based on the monoclonal antibody (mAb) 3B3 against S-adenosyl-L-homocysteine (SAH). The 50% inhibitory concentration (IC50) for this mAb against SAH was 2.94 nM with a linear range of 0.73-11.75 nM. The developed LFIA could detect SAH with a vLOD and cLOD of 10 nM and 2.07 nM, respectively. For tHcy detection in serum samples, the LFIA strip was able to detect tHcy after enzymatic hydrolysis, and the results correlated well with that of an enzymatic cycling test kit.

Colorimetric and Photoluminescent Probes Based on Iridium(Iii) Complexes for Highly Selective Detection of Homocysteine

Colorimetric and Photoluminescent Probes Based on Iridium(Iii) Complexes for Highly Selective Detection of Homocysteine

Analysis of homocysteine and thiol-containing compounds using the heterocyclic disulfides

Biological low-molecular aminothiols, such as homocysteine, are characterized by the weak absorption in the UV – visible range. Therefore, aminothiols analysis requires using thiol-specific chemosensors or derivatization procedure. Heterocyclic disulfides belong to a large group of derivatizing agents. However, the disulfides widely used in the analytical practice, such as 5,5'-dithiobis-(2-nitrobenzoic acid), have several disadvantages: limited acceptable pH range, low sensitivity, susceptibility to hydrolisys. Hence, the search for the effective derivatizing agents continues. In the current work, 2,2'-dithiobis[benzoxazole] and 8,8'-dithiobis-quinoline disulfides were used for the analysis of homocysteine. Precise and sensitive methods (homocysteine detection limit in the range of 1.1·10-6–7.8·10-6 M, relative standard deviation in the range of 1.5–4.6%) for the homocysteine determination have been developed, including spectrophotometric method, capillary electrophoresis (CE), high-performance liquid chromatography (HPLC) and kinetic method with the spectrophotometric detection. The proposed disulfides have also been successfully used in the analysis of sulfhydryl groups content in the sample of human urine.

Assembly of a Nanogold-Assisted Aptamer Sensor for Highly Sensitive Detection of Homocysteine

Assembly of a Nanogold-Assisted Aptamer Sensor for Highly Sensitive Detection of Homocysteine

Design and synthesis of poly-L-lysine-functionalized graphene quantum dots sensor for specific detection of cysteine and homocysteine

In this paper, a novel poly-l-lysine (PLL) surface functionalized graphene quantum dots (GQDs) based sensor was developed for detection of cysteine (cys) and homo cysteine (hcys). A fluorescent probe (PLL-GQDs) was then fabricated by surface functionalizing GQDs with PLL, a biodegradable polycationic electrolyte to improve the sensitivity and selectivity towards cys and hcys. The detection was based on the specific binding of cys and hcys to PLL at the PLL-GQDs surfaces, which enabled dynamic quenching via electrostatic and hydrophobic interactions. This fluorescent probe provided good linearity for the tested biothiols, ranging from 0 to 150 nM for cys, from 0 to 100 nM for hcys, with limit of detections (LODs) of 2.38 and 1.94 nM, respectively in BPS (pH 7.4). Interestingly, fabricated probe was also able to display a significant selectivity towards cys and hcys against known interfering molecules. The cytotoxicity study confirmed the biocompatibility of PLL-GQDs, enabling its future scope for cell adhesion and other biomedical applications. Besides, confocal study revealed the excellent penetrations of PLL-GQDs into cell cytoplasm and nucleus that validate the practical application of developed probe to detect cys and hcys at cellular level. The method was successfully applied for detection of cys and hcys in human serum sample. We expect the design concept presented here would be broadly used for selective and sensitive estimation of cys and hcys.

A Ratiometric, Fast-Responsive, and Single-Wavelength Excited Fluorescent Probe for the Discrimination of Cys and Hcy

The ratiometric detection of cysteine (Cys) and homocysteine (Hcy) is very challenging because of their highly similar chemical structures and properties. By introducing the phenylethynyl group into a coumarin dye as the sensing group, the ratiometric fluorescent probe CP was developed to selectively and rapidly discriminate between Cys and Hcy. With a single-wavelength excitation, the presence of Cys or Hcy induced the probe to produce distinct ratiometric fluorescence changes: from red (λmaxem = 608 nm) to blue (λmaxem = 485 nm) toward Cys and from red to mixed red/blue toward Hcy. Moreover, the probe was capable of visualizing and discriminating between intracellular Cys and Hcy in HeLa cells and zebrafish by exhibiting different ratiometric fluorescence signals.

Development of Second-Tier Liquid Chromatography-Tandem Mass Spectrometry Analysis for Expanded Newborn Screening in Japan.

To minimize false-positive cases in newborn screening by tandem mass spectrometry in Japan, practical second-tier liquid chromatography-tandem mass spectrometry analyses have been developed using a multimode ODS column with a single set of mobile phases and different gradient elution programs specific to the analysis of acylcarnitines, acylglycines, amino acids, and organic acids. Most analyses were performed using underivatized samples, except for analysis of methylcitric acid, and careful conditioning of the column was necessary for analyses of organic acids. Our second-tier tests enabled us to measure many metabolites useful for detection of target disorders, including allo-isoleucine, homocysteine, methylmalonic acid, and methylcitric acid. We found that accumulation of 3-hydroxyglutaric acid was specific to glutaric acidemia type I and that the ratio of 3-hydroxyisovaleric acid to 3-hydroxyisovalerylcarnitine was useful to detect newborns of mothers with 3-methylcrotonyl-CoA carboxylase deficiency. Data from the analysis of short-chain acylcarnitine and acylglycine were useful for differential diagnosis in cases positive for C5-OH-acylcarnitine or C5-acylcarnitine.