The effects of mouthwashes in human gingiva epithelial progenitor (HGEPp) cells.

OBJECTIVES: The gingiva epithelium accounts for a significant proportion of the surface around the tooth. An inflammatory reaction occurs in the presence of bacterial biofilm, adhesion is reduced, and the depth of the sulcus gingivalis increases. The most common antiseptic agents in oral rinses are chlorhexidine digluconate (CHX) and cetylpyridinium chloride. We examined long-lasting effects of residual concentrations of eight commercially available rinses. Our main goals were (i) to analyze the effect of different chemical compositions on cell proliferation, (ii) to examine apoptosis, and (iii) cell morphology on human epithelial progenitor cell line (HGEPp). MATERIALS AND METHODS: Cell proliferation was measured in a real-time system (0-48 h) by impedimetry (xCELLigence). Apoptosis was measured with labeled Annexin-V (BD-FACScalibur). RESULTS: Changes in proliferation were measured at certain concentrations: (i) H2O2 proved to be cytotoxic at almost all concentrations; (ii) low concentrations of CHX (0.0001%; 0.0003%) were proliferation inducers, while higher concentrations were cytotoxic; (iii) for ClO2, advantageous proliferative effect was observed over a broad concentration range (0.06-6 ppm). In mouthwashes, additives in the formulation (e.g., allantoin) appeared to influence cellular responses positively. Apoptosis marker assay results suggested a low-level activation by the tested agents. CONCLUSIONS: Mouthwashes and their reference compounds proved to have concentration-dependent cytotoxic effects on human gingival epithelial cells. CLINICAL RELEVANCE: A better understanding of the effects of mouthwashes and their reference compounds is particularly important. These concentration-dependent effects (cytotoxic or proliferation inducing) interfere with human cells physiology while being used in the fight against the pathogenic flora.

Apoptotic Effects of Drug Targeting Conjugates Containing Different GnRH Analogs on Colon Carcinoma Cells.

The wide range of cellular target reactions (e.g., antitumor) of gonadotropin-releasing hormone (GnRH) variants provides the possibility to develop multifunctional GnRH conjugates. The aim of our work was to compare the cytotoxic/apoptotic activity of different GnRH-based, daunorubicin (Dau)-linked conjugates with or without butyrated Lys in position 4 (4Lys(Bu)) at a molecular level in a human colorectal carcinoma cell line. Cell viability was measured by impedimetry, cellular uptake and apoptosis were studied by flow cytometry, and the expression of apoptosis-related genes was analyzed by qRT-PCR. The modification with 4Lys(Bu) resulted in an increased cytotoxic and apoptotic effects and cellular uptake of the GnRH-I and GnRH-III conjugates. Depending on the GnRH isoform and the presence of 4Lys(Bu), the conjugates could regulate the expression of several apoptosis-related genes, especially tumor necrosis factor (TNF), tumor protein p53 (TP53) and the members of growth-factor signaling. The stronger cytotoxicity of GnRH-I and GnRH-III conjugates containing 4Lys(Bu) was associated with a stronger inhibitory effect on the expression of growth-factor signaling elements in comparison with their 4Ser counterparts, in which the upregulation of TP53 and caspases (e.g., CASP9) seemed to play a more important role. We were able to provide further evidence that targeting the GnRH receptor could serve as a successful therapeutic approach in colon cancer, and GnRH-III-[4Lys(Bu),8Lys(Dau=Aoa)] proved to be the best candidate for this purpose.

Kynurenic acid and its derivatives are able to modulate the adhesion and locomotion of brain endothelial cells.

The neuroprotective actions of kynurenic acid (KYNA) and its derivatives in several neurodegenerative disorders [characterized by damage to the cerebral endothelium and to the blood-brain barrier (BBB)] are well established. Cell-extracellular matrix (ECM) adhesion is supposedly involved in recovery of impaired cerebral endothelium integrity (endothelial repair). The present work aimed to investigate the effects of KYNA and its synthetic derivatives on cellular behaviour (e.g. adhesion and locomotion) and on morphology of the GP8 rat brain endothelial cell line, modeling the BBB endothelium. The effects of KYNA and its derivatives on cell adhesion were measured using an impedance-based technique, the xCELLigence SP system. Holographic microscopy (Holomonitor™ M4) was used to analyse both chemokinetic responses and morphometry. The GP8 cells proved to be a suitable model cell line for investigating cell adhesion and the locomotion modulator effects of kynurenines. KYNA enhanced cell adhesion and spreading, and also decreased the migration/motility of GP8 cells at physiological concentrations (10-9 and 10-7 mol/L). The derivatives containing an amide side-chain at the C2 position (KYNA-A1 and A2) had lower adhesion inducer effects compared to KYNA. All synthetic analogues (except KYNA-A5) had a time-dependent inhibitory effect on GP8 cell adhesion at a supraphysiological concentration (10-3 mol/L). The immobilization promoting effect of KYNA and the adhesion inducer activity of its derivatives indicate that these compounds could contribute to maintaining or restoring the protective function of brain endothelium; they also suggest that cell-ECM adhesion and related cell responses (e.g. migration/motility) could be potential new targets of KYNA.

Drug targeting to decrease cardiotoxicity - determination of the cytotoxic effect of GnRH-based conjugates containing doxorubicin, daunorubicin and methotrexate on human cardiomyocytes and endothelial cells.

Background: Cardiomyopathy induced by the chemotherapeutic agents doxorubicin and daunorubicin is a major limiting factor for their application in cancer therapy. Chemotactic drug targeting potentially increases the tumor selectivity of drugs and decreases their cardiotoxicity. Increased expression of gonadotropin-releasing hormone (GnRH) receptors on the surface of tumor cells has been reported. Thus, the attachment of the aforementioned chemotherapeutic drugs to GnRH-based peptides may result in compounds with increased therapeutic efficacy. The objective of the present study was to examine the cytotoxic effect of anticancer drug-GnRH-conjugates against two essential cardiovascular cell types, such as cardiomyocytes and endothelial cells. Sixteen different previously developed GnRH-conjugates containing doxorubicin, daunorubicin and methotrexate were investigated in this study. Their cytotoxicity was determined on primary human cardiac myocytes (HCM) and human umbilical vein endothelial cells (HUVEC) using the xCELLigence SP system, which measures impedance changes caused by adhering cells on golden electrode arrays placed at the bottom of the wells. Slopes of impedance-time curves were calculated and for the quantitative determination of cytotoxicity, the difference to the control was analysed. Results: Doxorubicin and daunorubicin exhibited a cytotoxic effect on both cell types, at the highest concentrations tested. Doxorubicin-based conjugates (AN-152, GnRH-III(Dox-O-glut), GnRH-III(Dox-glut-GFLG) and GnRH-III(Dox=Aoa-GFLG) showed the same cytotoxic effect on cardiomyocytes. Among the daunorubicin-based conjugates, [4Lys(Ac)]-GnRH-III(Dau=Aoa), GnRH-III(Dau=Aoa-YRRL), {GnRH-III(Dau=Aoa-YRRL-C)}2 and {[4N-MeSer]-GnRH-III(Dau-C)}2 had a significant but decreased cytotoxic effect, while the other conjugates - GnRH-III(Dau=Aoa), GnRH-III(Dau=Aoa-K(Dau=Aoa)), [4Lys(Dau=Aoa)]-GnRH-III(Dau=Aoa), GnRH-III(Dau=Aoa-GFLG), {GnRH-III(Dau-C)}2 and [4N-MeSer]-GnRH-III(Dau=Aoa) - exerted no cytotoxic effect on cardiomyocytes. Mixed conjugates containing methotrexate and daunorubicin - GnRH-III(Mtx-K(Dau=Aoa)) and [4Lys(Mtx)]-GnRH-III(Dau=Aoa) - showed no cytotoxic effect on cardiomyocytes, as well. Conclusion: Based on these results, anticancer drug-GnRH-based conjugates with no cytotoxic effect on cardiomyocytes were identified. In the future, these compounds could provide a more targeted antitumor therapy with no cardiotoxic adverse effects. Moreover, impedimetric cytotoxicity analysis could be a valuable technique to determine the effect of drugs on cardiomyocytes.

Comparative cell biological study of in vitro antitumor and antimetastatic activity on melanoma cells of GnRH-III-containing conjugates modified with short-chain fatty acids.

Background: Peptide hormone-based targeted tumor therapy is an approved strategy to selectively block the tumor growth and spreading. The gonadotropin-releasing hormone receptors (GnRH-R) overexpressed on different tumors (e.g., melanoma) could be utilized for drug-targeting by application of a GnRH analog as a carrier to deliver a covalently linked chemotherapeutic drug directly to the tumor cells. In this study our aim was (i) to analyze the effects of GnRH-drug conjugates on melanoma cell proliferation, adhesion and migration, (ii) to study the mechanisms of tumor cell responses, and (iii) to compare the activities of conjugates with the free drug. Results: In the tested conjugates, daunorubicin (Dau) was coupled to 8Lys of GnRH-III (GnRH-III(Dau=Aoa)) or its derivatives modified with 4Lys acylated with short-chain fatty acids (acetyl group in [4Lys(Ac)]-GnRH-III(Dau=Aoa) and butyryl group in [4Lys(Bu)]-GnRH-III(Dau=Aoa)). The uptake of conjugates by A2058 melanoma model cells proved to be time dependent. Impedance-based proliferation measurements with xCELLigence SP system showed that all conjugates elicited irreversible tumor growth inhibitory effects mediated via a phosphoinositide 3-kinase-dependent signaling. GnRH-III(Dau=Aoa) and [4Lys(Ac)]-GnRH-III(Dau=Aoa) were shown to be blockers of the cell cycle in the G2/M phase, while [4Lys(Bu)]-GnRH-III(Dau=Aoa) rather induced apoptosis. In short-term, the melanoma cell adhesion was significantly increased by all the tested conjugates. The modification of the GnRH-III in position 4 was accompanied by an increased cellular uptake, higher cytotoxic and cell adhesion inducer activity. By studying the cell movement of A2058 cells with a holographic microscope, it was found that the migratory behavior of melanoma cells was increased by [4Lys(Ac)]-GnRH-III(Dau=Aoa), while the GnRH-III(Dau=Aoa) and [4Lys(Bu)]-GnRH-III(Dau=Aoa) decreased this activity. Conclusion: Internalization and cytotoxicity of the conjugates showed that GnRH-III peptides could guard Dau to melanoma cells and promote antitumor activity. [4Lys(Bu)]-GnRH-III(Dau=Aoa) possessing the butyryl side chain acting as a "second drug" proved to be the best candidate for targeted tumor therapy due to its cytotoxicity and immobilizing effect on tumor cell spreading. The applicability of impedimetry and holographic phase imaging for characterizing cancer cell behavior and effects of targeted chemotherapeutics with small structural differences (e.g., length of the side chain in 4Lys) was also clearly suggested.

Aptamer-graphene oxide for highly sensitive dual electrochemical detection of Plasmodium lactate dehydrogenase.

A 90 mer ssDNA aptamer (P38) enriched against Plasmodium falciparum lactate dehydrogenase (PfLDH) through SELEX process was immobilized over glassy carbon electrode (GCE) using graphene oxide (GO) as an immobilization matrix, and the modified electrode was investigated for detection of PfLDH. The GO was synthesized from powdered pencil graphite and characterized by XRD based on the increased interlayer distance between graphitic layers from 0.345 nm for graphite to 0.829 nm for GO. The immobilization of P38 on GO was confirmed by ID/IG intensity ratio in Raman spectra where, the ratio were 0.67, 0.915, and 1.35 for graphite, GO and P38-GO, respectively. The formation of the P38 layer over GO-GCE was evident from an increase in the surface height in AFM analysis of the electrode from ∼3.5 nm for GO-GCE to ∼27 nm for P38-GO-GCE. The developed aptasensor when challenged with the target, a detection of as low as 0.5 fM of PfLDH was demonstrated. The specificity of the aptasensor was confirmed through a voltametric measurement at 0.65 V of the reduced co-factor generated from the PfLDH catalysis. Studies on interference from some common proteins, storage stability, repeatability and analysis of real samples demonstrated the practical application potential of the aptasensor.

Label-free versus conventional cellular assays: Functional investigations on the human histamine H1 receptor.

A set of histamine H1 receptor (H1R) agonists and antagonists was characterized in functional assays, using dynamic mass redistribution (DMR), electric cell-substrate impedance sensing (ECIS) and various signaling pathway specific readouts (Fura-2 and aequorin calcium assays, arrestin recruitment (luciferase fragment complementation) assay, luciferase gene reporter assay). Data were gained from genetically engineered HEK293T cells and compared with reference data from GTPase assays and radioligand binding. Histamine and the other H1R agonists gave different assay-related pEC50 values, however, the order of potency was maintained. In the luciferase fragment complementation assay, the H1R preferred ß-arrestin2 over ß-arrestin1. The calcium and the impedimetric assay depended on Gq coupling of the H1R, as demonstrated by complete inhibition of the histamine-induced signals in the presence of the Gq inhibitor FR900359 (UBO-QIC). Whereas partial inhibition by FR900359 was observed in DMR and the gene reporter assay, pertussis toxin substantially decreased the response in DMR, but increased the luciferase signal, reflecting the contribution of both, Gq and Gi, to signaling in these assays. For antagonists, the results from DMR were essentially compatible with those from conventional readouts, whereas the impedance-based data revealed a trend towards higher pKb values. ECIS and calcium assays apparently only reflect Gq signaling, whereas DMR and gene reporter assays appear to integrate both, Gq and Gi mediated signaling. The results confirm the value of the label-free methods, DMR and ECIS, for the characterization of H1R ligands. Both noninvasive techniques are complementary to each other, but cannot fully replace reductionist signaling pathway focused assays.

Targeted tumor therapy by Rubia tinctorum L.: analytical characterization of hydroxyanthraquinones and investigation of their selective cytotoxic, adhesion and migration modulator effects on melanoma cell lines (A2058 and HT168-M1).

BACKGROUND: Alizarin and purpurin are di- and trihydroxyanthraquinones derived from Rubia tinctorum L. Previous pharmacological studies have demonstrated that they exhibit certain degree of selective inhibitory effects towards cancer cells suggesting their application as a targeted drug for cancer. Our present work was aimed to investigate the suitability of hydroxyanthraquinones of Rubia tinctorum L. for targeted tumor therapy. The effects of alizarin, purpurin and an aqueous extract from transformed hairy root culture of Rubia tinctorum L. were examined on (1) cell proliferation, (2) apoptosis, (3) cell adhesion/morphology and (4) migration (chemotaxis, chemokinesis) of human melanoma cell lines (A2058, HT168-M1) and human fibroblast cells (MRC-5), as well as (5) the aqueous extract was analytically characterized. METHODS: The aqueous extract was prepared from R. tinctorum hairy root culture and qualitatively analyzed by HPLC and ESI-MS methods. The cell growth inhibitory activity of anthraquinones was evaluated by MTT-assay and by flow cytometry. The effect of anthraquinones on cell adhesion was measured by an impedance based technique, the xCELLigence SP. For the chemotaxis assay NeuroProbe(®) chamber was used. Computer based holographic microscopy was applied to analyze chemokinetic responses as well as morphometry. Statistical significance was determined by the one-way ANOVA test. RESULTS: In the aqueous extract, munjistin (Mr = 284, tR = 18.4 min) as a principal component and three minor anthraquinones (pseudopurpurin, rubiadin and nordamnacanthal) were identified. The purpurin elicited a stronger but not apoptosis-mediated antitumor effect in melanoma cells (A2058: 10(-6)-10(-5) M: 90.6-64.1 %) than in normal fibroblasts (10(-6)-10(-5) M: 97.6-84.8 %). The aqueous extract in equimolar concentrations showed the most potent cytotoxicity after 72 h incubation (A2058: 10(-6)-10(-5) M: 87.4-55.0 %). All tested substances elicited chemorepellent effect in melanoma cells, while in MRC-5 fibroblasts, only the alizarin exhibited such a repellent character. Indices of chemokinesis measured by holographic microscopy (migration, migration directness, motility and motility speed) were significantly enhanced by alizarin and purpurin as well, while morphometric changes were weak in the two melanoma cell lines. CONCLUSIONS: Our results highlight the effective and selective inhibitory activity of purpurin towards melanoma cells and its possible use as a targeted anticancer agent. The anthraquinones of the cytotoxic extract are suggested to apply in drug delivery systems as an anticancer drug.

Electrochemical biosensor for serogroup specific diagnosis of leptospirosis.

A problem with the current leptospirosis diagnostic methods is the low sensitivity and specificity during the acute phase of illness. Rapid point-of-care (POC) assays with minimal sample utilization and low cost are desired in clinical practice. Here, we report for the first time lipopolysaccharide (LPS) based electrochemical biosensor that offers a rapid, highly sensitive, serogroup specific diagnosis of leptospirosis during the acute stage of infection and also to distinguish from other flu like infections. The proposed sensor is fabricated by the immobilization of LPS onto dodecanethiol (DT) modified gold electrode. Monolayer of DT is attached through covalent bond (Au-S) interaction onto the gold electrode. Thus, leptospiral antibodies from the human serum samples bind to the LPS present on self-assembled monolayer (SAM) of DT and showed a higher RCT value compared to SAM. The detection limit of the developed LPS sensor is estimated to be 100 nM. This biosensor is the first electrochemical sensing platform used for detection of LPS from Leptospira spp. This method is completely a solution-based diagnostic method and therefore it is rapid, simple, and sensitive; thus establishing a key technology towards a useful POC diagnostic strategy in serogroup level and hence an alternative to MAT.

Effect of dental antiseptic agents on the viability of human periodontal ligament cells.

OBJECTIVES: We aimed to study whether or not various dental antiseptic agents affect the viability and proliferation of human periodontal ligament cells (PDLCs). MATERIALS AND METHODS: Human PDLCs were isolated from a total of 10 surgically extracted impacted third molars and were.cultured in-vitro. The cells were exposed to commonly used dental antiseptics, including chlorhexidine, cetylpyridinium chloride, triclosan, povidone-iodine and sodium bicarbonate for ultra-short-term (10, 20, 30 sec), short-term (10, 20, 30 min) and long-term (24, 48 h) at various concentrations. Cell morphology was observed with light microscopy. Cell viability was studied with impedimetric real-time xCELLigence and resazurin-based alamarBlue® assays. We used one-way ANOVA with Tukey's and Bonferroni test (p < 0.05) for statistical analysis. RESULTS: Both alamarBlue® and xCELLigence analysis results were in agreement that ultra-short-term contact with cetylpyridinium chloride ≥ 0.01 mg/ml, chlorhexidine ≥ 1 mg/ml, triclosan ≥ 1 mg/ml and povidone-iodine ≥ 1 mg/ml as well as long-term exposure to cetylpyridinium chloride ≥ 0.001 mg/ml, chlorhexidine ≥ 0.01 mg/ml, triclosan ≥ 1 mg/ml, povidone-iodine ≥ 1 mg/ml and sodium bicarbonate ≥ 10 mg/ml was able to reduce the viability of human PDLCs significantly. According to the half-maximal inhibitory concentration (IC50) the rank of cytotoxicity was cetylpyridinium chloride > chlorhexidine > triclosan > povidone-iodine > sodium bicarbonate. CONCLUSIONS: Our findings suggest that the tested antiseptic agents were cytotoxic to human PDLCs at lower than practically applied concentrations in dental interventions.

Molecularly imprinted polymer as a synthetic receptor mimic for capacitive impedimetric selective recognition of Escherichia coli K-12.

We fabricated an electrochemical molecularly imprinted polymer (MIP) chemosensor for rapid identification and quantification of E. coli strain using 2-aminophenyl boronic acid as the functional monomer. This strain is a modified Gram-negative strain of Escherichia coli bacterium, an ordinary human gut component. The E. coli strongly interacts with a boronic acid because of porous and flexible polymers of the cell wall. The SEM imaging showed that the bacteria template was partially entrapped within the polymeric matrix in a single step. Moreover, this imaging confirmed E. coli K-12 cell template extraction effectiveness. The prepared MIP determined the E. coli K-12 strain up to 2.9 × 104 cells mL-1. The interference study performed in the presence of E. coli variants expressing different surface appendages (type 1 fimbriae or Antigen 43 protein) or Shewanella oneidensis MR1, another Gram-negative bacteria, demonstrated that the bacterial surface composition notably impacts sensing properties of the bacteria imprinted polymer.

Impedimetric determination of Cs(I) using AuNPs@PoPD-DB24C8: A targeted molecular-scale perturbation.

Most attributes of the bulk materials, especially in the solid-state, are directly dictated by a manner by which the molecules are ordered. Thus, it is expected that the possibility of controlling these structural orders would allow predominating some particular physical properties. The methodology used in this work follows the molecular scale perturbation occurred by Cs+ ion within a ternary composite of dibenzo-24-crown-8 (DB24C8), poly ortho-phenylenediamine (PoPD) and gold nanoparticles (AuNPs). Hypothetically, two former substances were respectively employed as recognition element and conductive platform to establish a monolithic structure that resembles supramolecular synthon in solid-state. The third precursor was Au(III) that carries out a dual role including vulcanization of the polymeric units via creating quinoid rings and solid signal amplification by deposition of AuNPs at the welded points. This strategy affords an intertwined ternary composite in which the electronical properties of the system can be directly affected by lowest agitation sensed by the recognition element, DB24C8, making the supported transducer capable of monitoring trace amount of Cs+ ion by Faradaic impedance spectroscopy (FIS) and single-frequency measurements (SFM). The fabricated sensor showed a signal change against Cs+ ion over the linear range of 0.6-25.0 nM with a detection limit of 0.37 nM (S/N = 3). Density functional theory (DFT) studies were used to explore the possible recognition mechanism, by which the incorporation of Cs+ ion meaningfully dispersed the structural order of the ternary system.

Physical Structure and Electrochemical Response of Diamond-Graphite Nanoplatelets: From CVD Synthesis to Label-Free Biosensors.

Hybrid diamond-graphite nanoplatelet (DGNP) thin films are produced and applied to label-free impedimetric biosensors for the first time, using avidin detection as a proof of concept. The DGNPs are synthesized by microwave plasma chemical vapor deposition through H2/CH4/N2 gas mixtures in a reproducible and rapid single-step process. The material building unit consists of an inner two-dimensional-like nanodiamond with preferential vertical alignment covered by and covalently bound to nanocrystalline graphite grains, exhibiting {111}diamond||{0002}graphite epitaxy. The DGNP films' morphostructural aspects are of interest for electrochemical transduction, in general, and for Faradaic impedimetric biosensors, in particular, combining enhanced surface area for biorecognition element loading and facile Faradaic charge transfer. Charge transfer rate constants in phosphate buffer saline/[Fe(CN)6]4- solution are shown to increase up to 5.6 × 10-3 cm s-1 upon N2 addition to DGNP synthesis. For the impedimetric detection of avidin, biotin molecules are covalently bound as avidin specific recognition elements on (3-aminopropyl)triethoxysilane-functionalized DGNP surfaces. Avidin quantification is attained within the 10-1000 µg mL-1 range following a logarithmic dependency. The limits of detection and of quantitation are 1.3 and 6.4 µg mL-1 (19 and 93 nM), respectively, and 2.3 and 13.8 µg mL-1 (33 and 200 nM) when considering the nonspecific response of the sensors.

Sub-femtomolar detection of HIV-1 gene using DNA immobilized on composite platform reinforced by a conductive polymer sandwiched between two nanostructured layers: A solid signal-amplification strategy.

This study introduces a signal amplification strategy rely on incorporating a specific polymer film between two typical nanostructured layers, aiming to improve the electrical properties of the platform to be able to transduce small binding event through sub-femtomolar detection of HIV-1 gene at the surface of the constructed biosensing device. The proposed composite was arrayed based on a conductive layer consist of p-aminobenzoic acid (PABA) sandwiched between the electrochemically reduced graphene oxide (ERGO) as the sub-layer, and the gold nanoparticles (AuNPs) as the interfacial layer. We computationally explored that how the use of such design enables the platform to transduce small changes in the interfacial properties of the biosensor, caused by low concentrations of HIV-1 gene, without needing any amplification strategy. Furthermore, it was found that the loin PABA conductive polymer sandwiched between two nanostructure layers play an artwork-ensemble role, which resulted in a good signal repeatability and stability during the relatively long successive incubation and detection procedures. The justification of using such an array of conductive layers was established on the attaining extra low-level of detection limit. The observed performance for probe-DNA immobilized on glassy carbon electrode (GC) modified with ERGO/PABA/AuNPs compared to the GC electrode modified with ERGO/AuNPs inspired us to perform computational calculations, a hybrid of ab-initio and semi-empirical quantum mechanics methods, to discover its probable molecular-scale reasons. A rapid single frequency impedance measurement (SFIM) was also employed to remarkably reduce the measurement time and diminish the probable nonspecific impedance changes. The proposed biosensor was used to evaluate the DNA target over an extremely wide concentration range from 0.1 fM to 10 nM, with a detection limit of 37 aM (S/N = 3).

Facile Fabrication of Surface-Imprinted Macroporous Films for Chemosensing of Human Chorionic Gonadotropin Hormone.

We present an improved approach for the preparation of highly selective and homogeneous molecular cavities in molecularly imprinted polymers (MIPs) via the combination of surface imprinting and semi-covalent imprinting. Toward that, first, a colloidal crystal mold was prepared via the Langmuir-Blodgett (LB) technique. Then, human chorionic gonadotropin (hCG) template protein was immobilized on the colloidal crystal mold. Later, hCG derivatization with electroactive functional monomers via amide chemistry was performed. In a final step, optimized potentiostatic polymerization of 2,3'-bithiophene enabled depositing an MIP film as the macroporous structure. This synergistic strategy resulted in the formation of molecularly imprinted cavities exclusively on the internal surface of the macropores, which were accessible after dissolution of silica molds. The recognition of hCG by the macroporous MIP film was transduced with the help of electric transducers, namely, extended-gate field-effect transistors (EG-FET) and capacitive impedimetry (CI). These readout strategies offered the ability to create chemosensors for the label-free determination of the hCG hormone. Other than the simple confirmation of pregnancy, hCG assay is a common tool for the diagnosis and follow-up of ectopic pregnancy or trophoblast tumors. Concentration measurements with these EG-FET and CI-based devices allowed real-time measurements of hCG in the range of 0.8-50  and 0.17-2.0 fM, respectively, in 10 mM carbonate buffer (pH = 10). Moreover, the selectivity of chemosensors with respect to protein interferences was very high.

Platelet impedance adhesiometry: A novel technique for the measurement of platelet adhesion and spreading.

INTRODUCTION: Thrombogenesis plays an important role in today's morbidity and mortality. Antithrombotics are among the most frequently prescribed drugs. Thorough knowledge of platelet function is needed for optimal clinical care. Platelet adhesion is a separate subprocess of platelet thrombus formation; still, no well-standardized technique for the isolated measurement of platelet adhesion exists. Impedimetry is one of the most reliable, state-of-art techniques to analyze cell adhesion, proliferation, viability, and cytotoxicity. We propose impedimetry as a feasible novel method for the isolated measurement of 2 significant platelet functions: adhesion and spreading. METHODS: Laboratory reference platelet agonists (epinephrine, ADP, and collagen) were applied to characterize platelet functions by impedimetry using the xCELLigence SP system. Platelet samples were obtained from 20 healthy patients under no drug therapy. Standard laboratory parameters and clinical patient history were also analyzed. RESULTS: Epinephrine and ADP increased platelet adhesion in a concentration-dependent manner, while collagen tended to have a negative effect. Serum sodium and calcium levels and age had a negative correlation with platelet adhesion induced by epinephrine and ADP, while increased immunoreactivity connected with allergic diseases was associated with increased platelet adhesion induced by epinephrine and ADP. ADP increased platelet spreading in a concentration-dependent manner. CONCLUSION: Impedimetry proved to be a useful and sensitive method for the qualitative and quantitated measurement of platelet adhesion, even differentiating between subgroups of a healthy population. This novel technique is offered as an important method in the further investigation of platelet function.

A novel hydrogel scaffold for periodontal ligament stem cells.

Periodontal ligament stem cells (PDLSCs) possess extensive regeneration potential. However, their therapeutic application demands a scaffold with appropriate properties. HydroMatrix (HydM) is a novel injectable peptide nanofiber hydrogel developed recently for cell culture. Our aim was to test whether HydM would be a suitable scaffold for proliferation and osteogenic differentiation of PDLSCs. PDLSCs were seeded on non-coated or HydM-coated surfaces. Both real-time impedance analysis and cell viability assay documented cell growth on HydM. PDLSCs showed healthy, fibroblast-like morphology on the hydrogel. After a 3-week-long culture in osteogenic medium, mineralization was much more intense in HydM cultures compared to control. Alkaline phosphatase activity of the cells grown on the gels reached the non-coated control levels. Our data provided evidence that PDLSCs can adhere, survive, migrate, and proliferate on HydM and this gel also supports their osteogenic differentiation. We first applied impedimetry for dental stem cells cultured on a scaffold. HydM is ideal for in vitro studies of PDLSCs. It may also serve not only as a reference material but also in the future as a promising biocompatible scaffold for preclinical studies.

A new bifunctional nanostructure based on Two-Dimensional nanolayered of Co(OH)2 exfoliated graphitic carbon nitride as a high performance enzyme-less glucose sensor: Impedimetric and amperometric detection.

A novel non-enzymatic glucose sensor was constructed based on the nanolayered Co(OH)2 deposited on polymeric graphitic carbon nitride (Co(OH)2-g-C3N4) via chemical bath deposition. The two-dimensional nanocomposite was used to modify a carbon paste electrode and its electrochemical performance of electrode was carefully evaluated. The electrochemical oxidation of glucose at modified electrode was studied by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in 0.2 M KOH. It revealed a remarkable electrocatalytic behavior and noticeably synergistic effect as a result of decent exfoliation of graphitic carbon nitride in the presence of Co(OH)2. The amperometric response achieved by the modified electrode showed two calibration ranges with an excellent selectivity. A single-frequency impedance method was applied for glucose determination as an alternative to conventional EIS methods. The developed sensor represents a high sensitivity, wide concentration range (25 µM - 420.0 mM by impedimetry and 6.6-9800 µM by amperometry), and high reproducibility. The modified electrode showed a good selectivity for glucose over potentially interfering materials such as dopamine, ascorbic acid, urea, and uric acid. Finally, the Co(OH)2-g-C3N4-CPE was utilized for quantification of glucose in blood serum samples.

Programmed Transfer of Sequence Information into a Molecularly Imprinted Polymer for Hexakis(2,2'-bithien-5-yl) DNA Analogue Formation toward Single-Nucleotide-Polymorphism Detection.

A new strategy of simple, inexpensive, rapid, and label-free single-nucleotide-polymorphism (SNP) detection using robust chemosensors with piezomicrogravimetric, surface plasmon resonance, or capacitive impedimetry (CI) signal transduction is reported. Using these chemosensors, selective detection of a genetically relevant oligonucleotide under FIA conditions within 2 min is accomplished. An invulnerable-to-nonspecific interaction molecularly imprinted polymer (MIP) with electrochemically synthesized probes of hexameric 2,2'-bithien-5-yl DNA analogues discriminating single purine-nucleobase mismatch at room temperature was used. With density functional theory modeling, the synthetic procedures developed, and isothermal titration calorimetry quantification, adenine (A)- or thymine (T)-substituted 2,2'-bithien-5-yl functional monomers capable of Watson-Crick nucleobase pairing with the TATAAA oligodeoxyribonucleotide template or its peptide nucleic acid (PNA) analogue were designed. Characterized by spectroscopic techniques, molecular cavities exposed the ordered nucleobases on the 2,2'-bithien-5-yl polymeric backbone of the TTTATA hexamer probe designed to hybridize the complementary TATAAA template. In that way, an artificial TATAAA-promoter sequence was formed in the MIP. The purine nucleobases of this sequence are known to be recognized by RNA polymerase to initiate the transcription in eukaryotes. The hexamer strongly hybridized TATAAA with the complex stability constant KsTTTATA-TATAAA = ka/kd ≈ 106 M-1, as high as that characteristic for longer-chain DNA-PNA hybrids. The CI chemosensor revealed a 5 nM limit of detection, quite appreciable as for the hexadeoxyribonucleotide. Molecular imprinting increased the chemosensor sensitivity to the TATAAA analyte by over 4 times compared to that of the nonimprinted polymer. The herein-devised detection platform enabled the generation of a library of hexamer probes for typing the majority of SNP probes as well as studying a molecular mechanism of the complex transcription machinery, physics of single polymer molecules, and stable genetic nanomaterials.

Highly sensitive label free electrochemical detection of VGEF165 tumor marker based on "signal off" and "signal on" strategies using an anti-VEGF165 aptamer immobilized BSA-gold nanoclusters/ionic liquid/glassy carbon electrode.

In this work, a label free electrochemical aptasensor for the detection of ultra-traces of vascular endothelial growth factor (VEGF165) based on "signal off" and "signal on" mechanisms of response was developed. The BSA-gold nanoclusters/ionic liquid (BSA-AuNCs/IL) was used as a suitable nanocomposite platform for immobilization of the aptamer on a glassy carbon electrode. In "signal off" mechanism, the interaction of VEGF165 with its anti-VEGF165 aptamers, resulted in desorption of methylene blue (MB) probe from aptamer and its release into solution. Consequently, the decrease in current intensity of the differential pulse voltammogram of adsorbed MB was monitored and found to be linearly proportional with increasing concentration of VEGF165 in sample solution in the range of 1-120 pM with a limit of detection of 0.32p M. While, in "signal on" mechanism, the interaction of immobilized anti-VEGF165 aptamers on the electrode surface with VEGF165, led to more mass-transfer limiting of the [Fe(CN)6](3-/4-) probe to the electrode surface. Therefore, the charge transfer resistance (Rct) of the probe was increased linearly with increasing concentration of VEGF165 in the range of 2.5-250 pM with a limit of detection of 0.48 pM. The experimental results demonstrated that both of these mechanisms are suitable for determination of low levels of the VEGF165 tumor marker in serum samples.