Effective voltammetric tool for simultaneous detection of MMP-1, MMP-2, and MMP-9; important non-small cell lung cancer biomarkers.

Simultaneous detection of multiple biomarkers can allow to reduce the costs of medical diagnostics, and thus improve the accuracy and effectiveness of disease diagnosis and prognosis. Here, for the first time, we present a low-cost, simple, and rapid method for simultaneous detection of three matrix metalloproteinases (MMP-1, MMP-2, and MMP-9) that play important roles in the progression of lung cancer. The sensor matrix was constructed using a G2 polyamidoamine dendrimer (PAMAM) containing amino, carboxyl, and sulfhydryl groups. The recognition process was based on specific enzymatic cleavage of the Gly-Ile peptide bond by MMP-1, Gly-Leu bond by MMP-2, and Gly-Met bond by MMP-9, and monitoring was done by square wave voltammetry. The activity of metalloproteinases was detected based on the change of current signals of redox receptors (dipeptides labeled with electroactive compounds) covalently anchored onto the electrode surface. The conditions of the biosensor construction, including the concentration of receptors on the sensor surface and the time of interaction of the receptor with the analyte, were carefully optimized. Under optimal conditions, the linear response of the developed method ranged from 1.0⋅10-8 to 1.0 mg⋅L-1, and the limit of detection for MMP-1, MMP-2, and MMP-9 was 0.35, 0.62, and 1.10 fg⋅mL-1, respectively. The constructed biosensor enabled us to efficiently profile the levels of active forms of MMP-1, MMP-2, and MMP-9 in tissue samples (plasma and lung and tumor extracts). Thus, the developed biosensor can aid in the early detection and diagnosis of lung cancer.

A rapid, selective, and ultrasensitive voltammetric and gravimetric protocol for MMP-1 active form detection.

In this paper a rapid, selective, and ultrasensitive protocol for the detection of the active form of matrix metalloproteinase-1 (MMP-1), which is a novel predictive and prognostic biomarker, was presented, which might strengthen the current predictive systems. The biosensor construction procedure was extremely simple, economical, and time-saving, as it involved only the chemisorption step of the voltammetrically active receptor (tripeptide (Cys-Gly-Ile) labeled with methylene blue (MB) and the sealing thiol. The active form of MMP-1 was recognized based on its hydrolytic activity; as a consequence, the receptor fragment (-Ile-MB) was removed from the sensor surface. The biosensors constructed were characterized by a wide dynamic concentration response range (1.0 pg mL-1-1.0 µg mL-1) and a low detection limit (33 fg mL-1), especially the biosensor with voltammetric detection, without the amplification step. One of the important advantages of the proposed biosensors is that they can be directly used to analyze the content of the active form of MMP-1 in clinical samples without the dilution step and any other preparation step.

Poly(amidoamine) dendrimer immunosensor for ultrasensitive gravimetric and electrochemical detection of matrix metalloproteinase-9.

Monitoring the level of matrix metalloproteinase-9 (MMP-9) and inhibiting its expression is important for the diagnosis and treatment of various diseases. However, the analysis of MMP-9 is challenging owing to its very low content in the blood, especially at the early stages of diseases. Therefore, we developed an ultrasensitive and easy-to-use immunosensor based on a three-dimensional (3D) bioplatform for the determination of the total MMP-9 concentration in plasma. The used 3D bioplatform (G2 poly(amidoamine) dendrimer; PAMAM) improved the sensitivity of the determination by significantly expanding the surface area of the receptor layer. The antigen-antibody recognition process was controlled by quartz crystal microbalance with dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS). The effect of the orientation of antibody molecules in the sensing layer on the work parameters of the immunosensor was analyzed using unmodified PAMAM (PAMAM-NH2) and PAMAM functionalized with -COOH groups (PAMAM-COOH). The developed immunosensor based on PAMAM-NH2 was characterized by a lower detection limit (LOD = 2.0 pg⋅mL-1) and wider analytical range (1·10-4 - 5 µg⋅mL-1 for EIS and QCM-D) compared to PAMAM-COOH immunosensor (EIS: 1·10-4 - 0.5 µg⋅mL-1; QCM-D: 5·10-4 - 0.5 µg⋅mL-1). The functionality of the proposed device was verified in spiked plasma. The recoveries determined in commercial human and rat plasma and noncommercial rat plasma were very close to the value of 100% and in the range of 96-120% for Au/PAMAM-NH2/Ab and Au/PAMAM-COOH/Ab immunosensors, respectively. The designed analytical devices showed high selectivity and sensitivity without the use of any amplifiers such as metal nanoparticles or enzymes.

Novel electrogravimetric biosensors for the ultrasensitive detection of plasma matrix metalloproteinase-2 considered a potential tumor biomarker.

In this study, we developed novel, simple gravimetric and voltammetric sensors for the ultrasensitive detection of active matrix metalloproteinase (MMP)-2 in plasma. The developed sensors are cost-effective, require a very less amount of reagents, and are time-saving. They detect MMP-2 based on antigen-antibody recognition and its ability to cleave glycine-leucine peptide bond. The three-dimensional bioplatform of the sensors consisted of a cationic polyethyleneimine (PEI) polymer that facilitated robust immobilization of the dipeptide labeled with anthraquinone (AQ), or antibody molecules in appropriate density, which was crucial for biosensing. Detection was performed using quartz crystal microbalance with dissipation and voltammetry. The results showed that the developed sensors were characterized by high stability, wide analytical range (2.0 pg mL-1 to 5.0 µg mL-1), and low detection limit (ca. 10 fg mL-1). They also exhibited excellent efficiency in the determination of active MMP-2 in real samples, such as blood plasma. The developed sensors may hold great promise for the early diagnosis of cancers.

Enzymatic cleavage of specific dipeptide conjugated with ferrocene as a flexible ultra-sensitive and fast voltammetric assay of matrix metalloproteinase-9 considered a prognostic cancer biomarker in plasma samples.

Studies over the last decade have shown that matrix metalloproteinases (MMPs) play a key role in the growth and metastasis of cancer. This zinc-dependent family of endopeptidases is crucial for the degradation of extracellular matrix (ECM), as well as serves as important ECM transducers which have been recognized as early biomarkers for both cancer diagnosis and treatment. In this study, we designed a new type of voltammetric biosensor, composed of a glycine-methionine dipeptide conjugated covalently to ferrocene (Gly-Met-Fc), for fast and ultrasensitive detection of the active form of MMP-9 in plasma samples. The detection was based on specific enzymatic cleavage of the Gly-Met peptide bond, which was monitored by voltammetry and gravimetry measurements. The ferrocene units act as voltammetric visualizers for the detection process. The cysteamine layer directly anchored to the gold surface ensured that the packing density of Gly-Met-Fc in the receptor layer was appropriate for the sensitive detection of MMP-9 in its active form. The developed biosensor was characterized by the widest analytical range (2.0·10-6 - 5.0 µg⋅mL-1) and low detection limit (0.04 pg⋅mL-1). Another valuable feature of the proposed biosensor is that it can be applied directly to the plasma samples without any additional preparation step and thus speeds up the analysis.

Electrochemical Recognition of Aromatic Species with Ferrocenylated 1,3,5-Triazine- or 1,3,5-Triphenylbenzene-Containing Highly Organized Molecules.

Two ferrocenylated organized molecules comprising 1,3,5-triphenylbenzene (Fc-1) or 2,4,6-triphenyl-1,3,5-triazine skeletons (Fc-2) were used for the first time as receptor layers for the electrochemical recognition of polycyclic aromatic hydrocarbons. While our group recently reported the synthesis of Fc-1, herein the facile synthesis of its 2,4,6-triphenyl-1,3,5-triazine-containing structural analog (Fc-2) is presented. Although the synthesis of Fc-2 was found to be challenging, we achieved a very high yield (89 %) under mild conditions using an acid-catalyzed imine-bond formation reaction in 1,4-dioxane:toluene solvent system. Title compounds were comprehensively characterized with various analytical techniques, including spectroscopic (NMR, FT-IR, FT-Raman spectroscopy) methods, high-resolution mass spectrometry (HRMS), microscopic (SEM) and electrochemical (CV) analyses. Fc-1 and Fc-2 were also used for the construction of the first-of-a-kind recognition layers (electrochemical sensors) dedicated to the recognition of polycyclic aromatic hydrocarbons. Fully constructed innovative sensors enabled the efficient recognition of analytes since the limit of detection (LOD) values were not higher than 2.9 µM. Comparative studies between the working parameters of electrochemical sensors comprising Fc-1 or Fc-2 were also included in this work.

Tracking of Glycans Structure and Metallomics Profiles in BRAF Mutated Melanoma Cells Treated with Vemurafenib.

Nearly half of patients with advanced and metastatic melanomas harbor a BRAF mutation. Vemurafenib (VEM), a BRAF inhibitor, is used to treat such patients, however, responses to VEM are very short-lived due to intrinsic, adaptive and/or acquired resistance. In this context, we present the action of the B-Raf serine-threonine protein kinase inhibitor (vemurafenib) on the glycans structure and metallomics profiles in melanoma cells without (MeWo) and with (G-361) BRAF mutations. The studies were performed using α1-acid glycoprotein (AGP), a well-known acute-phase protein, and concanavalin A (Con A), which served as the model receptor. The detection of changes in the structure of glycans can be successfully carried out based on the frequency shifts and the charge transfer resistance after interaction of AGP with Con A in different VEM treatments using QCM-D and EIS measurements. These changes were also proved based on the cell ultrastructure examined by TEM and SEM. The LA-ICP-MS studies provided details on the metallomics profile in melanoma cells treated with and without VEM. The studies evidence that vemurafenib modifies the glycans structures and metallomics profile in melanoma cells harboring BRAF mutation that can be further implied in the resistance phenomenon. Therefore, our data opens a new avenue for further studies in the short-term addressing novel targets that hopefully can be used to improve the therapeutic regiment in advanced melanoma patients. The innovating potential of this study is fully credible and has a real impact on the global patient society suffering from advanced and metastatic melanomas.

A chromatography-free total synthesis of a ferrocene-containing dendrimer exhibiting the property of recognizing 9,10-diphenylanthracene.

Molecules comprising several ferrocene residues constitute an intriguing group of compounds for various applications. Here, the total synthesis of a new example of a ferrocene-containing dendrimer is presented. The target compound was obtained in excellent combined yield (65%) employing facile, chromatography-free methods at each step. Interesting findings, meeting the dynamic covalent chemistry concept, are reported. Cyclic voltammetry analyses revealed one pair of current signals for the ferrocene moieties. Ultimately, the synthesized ferrocene-containing dendrimer has been used as an innovative recognition material for 9,10-diphenylanthracene, a polycyclic aromatic hydrocarbon, with the limit of detection value equal to 0.06 µM.