Simplified Synthesis and Stability Assessment of Aflatoxin B1-Lysine and Aflatoxin G1-Lysine

Aflatoxins B1 (AFB1) and G1 (AFG1) are carcinogenic mycotoxins that contaminate crops such as maize and groundnuts worldwide. The broadly accepted method to assess chronic human aflatoxin exposure is by quantifying the amount of aflatoxin adducted to human serum albumin. This has been reported using ELISA, HPLC, or LC-MS/MS to measure the amount of AFB1-lysine released after proteolysis of serum albumin. LC-MS/MS is the most accurate method but requires both isotopically labelled and unlabelled AFB1-lysine standards, which are not commercially available. In this work, we report a simplified synthetic route to produce unlabelled, deuterated and 13C6 15N2 labelled aflatoxin B1-lysine and for the first-time aflatoxin G1-lysine. Additionally, we report on the stability of these compounds during storage. This simplified synthetic approach will make the production of these important standards more feasible for laboratories performing aflatoxin exposure studies.

New Candidates for Biomarkers and Drug Targets of Ischemic Stroke—A First Dynamic LC-MS Human Serum Proteomic Study

(1) Background: The aim of this dynamic-LC/MS-human-serum-proteomic-study was to identify potential proteins-candidates for biomarkers of acute ischemic stroke, their changes during acute phase of stroke and to define potential novel drug-targets. (2) Methods: A total of 32 patients (29–80 years) with acute ischemic stroke were enrolled to the study. The control group constituted 29 demographically-matched volunteers. Subjects with stroke presented clinical symptoms lasting no longer than 24 h, confirmed by neurological-examination and/or new cerebral ischemia visualized in the CT scans (computed tomography). The analysis of plasma proteome was performed using LC-MS (liquid chromatography–mass spectrometry). (3) Results: Ten proteins with significantly different serum concentrations between groups volunteers were: complement-factor-B, apolipoprotein-A-I, fibronectin, alpha-2-HS-glycoprotein, alpha-1B-glycoprotein, heat-shock-cognate-71kDa protein/heat-shock-related-70kDa-protein-2, thymidine phosphorylase-2, cytoplasmic-tryptophan-tRNA-ligase, ficolin-2, beta-Ala-His-dipeptidase. (4) Conclusions: This is the first dynamic LC-MS study performed on a clinical model which differentiates serum proteome of patients in acute phase of ischemic stroke in time series and compares to control group. Listed proteins should be considered as risk factors, markers of ischemic stroke or potential therapeutic targets. Further clinical validation might define their exact role in differential diagnostics, monitoring the course of the ischemic stroke or specifying them as novel drug targets.

An Ultra-Sensitive Molecularly Imprinted Poly(Aniline) Based Electrochemical Sensor for the Determination of Bisphenol A in Synthetic Human Serum Specimen and Plastic Bottled Water Samples

We explain the development of an ultra-sensitive molecularly imprinted polymer-based electrochemical sensor for rapid and selective determination of bisphenol A (BPA) in human serum and water samples. Electropolymerization of functional monomer aniline was performed in the presence of BPA by cyclic voltammetry (CV) to prepare a molecularly imprinted poly(aniline) based GCE sensor (MIP(ANI)/GCE). The developed MIP surface was characterized using Fourier-transform infrared spectroscopy, Raman spectrometry, scanning electron microscopy, contact angle measurements, CV, and electrochemical impedance spectroscopy. The MIP(ANI)/GCE sensor showed a highly sensitive performance with a linear range of 1.0 and 8.0×10−15 M. The limit of detection (LOD) and limit of quantification (LOQ) values were 0.193 and 0.643 fM, respectively. The applicability of the MIP(ANI)/GCE was assessed by applying it to human serum and plastic bottled water samples. The LOD and LOQ values were calculated as 0.257 and 0.856 fM for the serum sample. Imprinting factor and interference studies were also carried out using similarly structured compounds and the most common interfering agents showing the selectivity of the MIP(ANI)/GCE sensor. Finally, the non-imprinted polymer (NIP)-based sensor was prepared to control the MIP(ANI)/GCE performance.

Development of Cobalt-Binding Peptide Chelate from Human Serum Albumin: Cobalt-Binding Properties and Stability

Radioactive isotopes are used as drugs or contrast agents in the medical field after being conjugated with chelates such as DOTA, NOTA, DTPA, TETA, CyDTA, TRITA, and DPDP. The N-terminal sequence of human serum albumin (HSA) is known as a metal binding site, such as for Co2+, Cu2+, and Ni2+. For this study, we designed and synthesized wAlb12 peptide from the N-terminal region of HSA, which can bind to cobalt, to develop a peptide-based chelate. The wAlb12 with a random coil structure tightly binds to the Co(II) ion. Moreover, the binding property of wAlb12 toward Co(II) was confirmed using various spectroscopic experiments. To identify the binding site of wAlb12, the analogs were synthesized by alanine scanning mutagenesis. Among them, H3A and Ac-wAlb12 did not bind to Co(II). The analysis of the binding regions confirmed that the His3 and α-amino group of the N-terminal region are important for Co(II) binding. The wAlb12 bound to Co(II) with Kd of 75 μM determined by isothermal titration calorimetry when analyzed by a single-site binding model. For the use of wAlb12 as a chelate in humans, its cytotoxicity and stability were investigated. Trypsin stability showed that the wAlb12 − Co(II) complex was more stable than wAlb12 alone. Furthermore, the cell viability analysis showed wAlb12 and wAlb12 + Co(II) to be non-toxic to the Raw 264.7 and HEK 293T cell lines. Therefore, a hot radioactive isotope such as cobalt-57 will have the same effect as a stable isotope cobalt. Accordingly, we expect wAlb12 to be used as a peptide chelate that binds with radioactive isotopes.