Reduced graphene-oxide transducers for biosensing applications beyond the Debye-screening limit.

In the field of label-free biosensing, various transducer materials and strategies are under investigation to overcome the Debye-screening limitation of charged biomolecules. We demonstrate an in-line, impedimetric aptasensor with reduced graphene-oxide (rGO) thin films as transducers to detect prostate specific antigens (PSA) in a physiological buffer solution. Unlike classical electrochemical impedance spectroscopy (EIS), this direct, label-free and fully-electronic biosensor approach does not need any redox markers. As specific capture molecules, short anti-PSA aptamers ensured a close binding of the target molecules to the transducer surfaces. Results showed a limit of detection smaller than 33 pM of PSA and a wide detection range from 0.033 to 330 nM fully covering the clinically relevant range of PSA (0.115-0.290 nM). This promising performance can be attributed to the bipolar electronic transport characteristics of the ultra-thin rGO layers similar to pristine graphene. The attachment of target biomolecules to the films changes the resistance of the rGO thin films. Such an in-line EIS configuration with rGO thin films opens promising prospects for biosensing beyond the Debye-screening limitation, which is a major challenge for conventional semiconductor field-effect devices towards clinical applications.

Selection of Aptamers Against Whole Living Cells: From Cell-SELEX to Identification of Biomarkers.

Aptamer selection protocols, named cell-SELEX, have been developed to target trans-membrane proteins using whole living cells as target. This technique presents several advantages. (1) It does not necessitate the use of purified proteins. (2) Aptamers are selected against membrane proteins in their native conformation. (3) Cell-SELEX can be performed to identify aptamers against biomarkers differentially expressed between different cell lines without prior knowledge of the targets. (4) Aptamers identified by cell-SELEX can be further used to purify their targets and to identify new biomarkers. Here, we provide a protocol of cell-SELEX including the preparation of an oligonucleotide library, next-generation sequencing and radioactive binding assays. Furthermore, we also provide a protocol to purify and identify the target of these aptamers. These protocols could be useful for the discovery of lead therapeutic compounds and diagnostic cell-surface biomarkers.

Highly sensitive dual mode electrochemical platform for microRNA detection.

MicroRNAs (miRNAs) play crucial regulatory roles in various human diseases including cancer, making them promising biomarkers. However, given the low levels of miRNAs present in blood, their use as cancer biomarkers requires the development of simple and effective analytical methods. Herein, we report the development of a highly sensitive dual mode electrochemical platform for the detection of microRNAs. The platform was developed using peptide nucleic acids as probes on gold electrode surfaces to capture target miRNAs. A simple amplification strategy using gold nanoparticles has been employed exploiting the inherent charges of the nucleic acids. Electrochemical impedance spectroscopy was used to monitor the changes in capacitance upon any binding event, without the need for any redox markers. By using thiolated ferrocene, a complementary detection mode on the same sensor was developed where the increasing peaks of ferrocene were recorded using square wave voltammetry with increasing miRNA concentration. This dual-mode approach allows detection of miRNA with a limit of detection of 0.37 fM and a wide dynamic range from 1 fM to 100 nM along with clear distinction from mismatched target miRNA sequences. The electrochemical platform developed can be easily expanded to other miRNA/DNA detection along with the development of microarray platforms.

Electrochemical functionalization of polypyrrole through amine oxidation of poly(amidoamine) dendrimers: Application to DNA biosensor.

Electrochemical patterning method has been developed to fabricate composite based on polypyrrole (PPy) film and poly(amidoamine) dendrimers of fourth generation (PAMAM G4). PPy layer was generated using electrochemical polymerization of pyrrole on a gold electrode. PPy film was then modified with PAMAM G4 using amines electro-oxidation method. Covalent bonding of PAMAM G4 and the formation of PPy-PAMAM composite was characterized using Fourier Transform Infrared Spectroscopy (FT-IR) and X-ray Photoelectron Spectroscopy (XPS). Ferrocenyl groups were then attached to such surface as a redox marker. Electrochemical properties of the modified nanomaterial (PPy-PAMAM-Fc) were studied using both amperometric and impedimetric methods to demonstrate the efficiency of electron transfer through the modified PPy layer. The obtained electrical and electrochemical properties were compared to a composite where PPy bearing carboxylic acid functions was chemically modified with PAMAM G4 by covalent attachment through formation of amid bond (PPy-CONH-PAMAM). The above mentioned studies showed that electrochemical patterning does not disturb the electronic properties of PPy. The effect of the number of functional groups introduced by the electrochemical patterning was demonstrated through the association of various compounds (ethylenediamine, PAMAM G2 and PAMAM G6). We demonstrated that such compounds could be applied in the biosensors technology. The modified PPy-PAMAM-Fc was evaluated as a platform for DNA sensing. High performance in the DNA detection by variation of the electrochemical signal of ferrocene was obtained with detection limit of 0.4 fM. Furthermore, such approach of electrochemical patterning by oxidation of amines could be applied for chemical modification of PPy and open a new way in various biosensing application involving functionalized PPy.

Direct detection of OTA by impedimetric aptasensor based on modified polypyrrole-dendrimers.

Ochratoxin A (OTA) is a carcinogenic mycotoxin that contaminates food such as cereals, wine and beer; therefore it represents a risk for human health. Consequently, the allowed concentration of OTA in food is regulated by governmental organizations and its detection is of major agronomical interest. In the current study we report the development of an electrochemical aptasensor able to directly detect trace OTA without any amplification procedure. This aptasensor was constructed by coating the surface of a gold electrode with a film layer of modified polypyrrole (PPy), which was thereafter covalently bound to polyamidoamine dendrimers of the fourth generation (PAMAM G4). Finally, DNA aptamers that specifically binds OTA were covalently bound to the PAMAM G4 providing the aptasensor, which was characterized by using both Atomic Force Microscopy (AFM) and Surface Plasmon Resonance (SPR) techniques. The study of OTA detection by the constructed electrochemical aptasensor was performed using Electrochemical Impedance Spectroscopy (EIS) and revealed that the presence of OTA led to the modification of the electrical properties of the PPy layer. These modifications could be assigned to conformational changes in the folding of the aptamers upon specific binding of OTA. The aptasensor had a dynamic range of up to 5 µg L(-1) of OTA and a detection limit of 2 ng L(-1) of OTA, which is below the OTA concentration allowed in food by the European regulations. The efficient detection of OTA by this electrochemical aptasensor provides an unforeseen platform that could be used for the detection of various small molecules through specific aptamer association.

Optimisation and Characterisation of Anti-Fouling Ternary SAM Layers for Impedance-Based Aptasensors.

An aptasensor with enhanced anti-fouling properties has been developed. As a case study, the aptasensor was designed with specificity for human thrombin. The sensing platform was developed on screen printed electrodes and is composed of a self-assembled monolayer made from a ternary mixture of 15-base thiolated DNA aptamers specific for human thrombin co-immobilised with 1,6-hexanedithiol (HDT) and further passivated with 1-mercapto-6-hexanol (MCH). HDT binds to the surface by two of its thiol groups forming alkyl chain bridges and this architecture protects from non-specific attachment of molecules to the electrode surface. Using Electrochemical Impedance Spectroscopy (EIS), the aptasensor is able to detect human thrombin as variations in charge transfer resistance (Rct) upon protein binding. After exposure to a high concentration of non-specific Bovine Serum Albumin (BSA) solution, no changes in the Rct value were observed, highlighting the bio-fouling resistance of the surface generated. In this paper, we present the optimisation and characterisation of the aptasensor based on the ternary self-assembled monolayer (SAM) layer. We show that anti-fouling properties depend on the type of gold surface used for biosensor construction, which was also confirmed by contact angle measurements. We further studied the ratio between aptamers and HDT, which can determine the specificity and selectivity of the sensing layer. We also report the influence of buffer pH and temperature used for incubation of electrodes with proteins on detection and anti-fouling properties. Finally, the stability of the aptasensor was studied by storage of modified electrodes for up to 28 days in different buffers and atmospheric conditions. Aptasensors based on ternary SAM layers are highly promising for clinical applications for detection of a range of proteins in real biological samples.

E-DNA sensor of Mycobacterium tuberculosis based on electrochemical assembly of nanomaterials (MWCNTs/PPy/PAMAM).

Two-step electrochemical patterning methods have been employed to elaborate composite nanomaterials formed with multiwalled carbon nanotubes (MWCNTs) coated with polypyrrole (PPy) and redox PAMAM dendrimers. The nanomaterial has been demonstrated as a molecular transducer for electrochemical DNA detection. The nanocomposite MWCNTs-PPy has been formed by wrapping the PPy film on MWCNTs during electrochemical polymerization of pyrrole on the gold electrode. The MWCNTs-PPy layer was modified with PAMAM dendrimers of fourth generation (PAMAM G4) with covalent bonding by electro-oxidation method. Ferrocenyl groups were then attached to the surface as a redox marker. The electrochemical properties of the nanomaterial (MWCNTs-PPy-PAMAM-Fc) were studied using both square wave voltammetry and cyclic voltammetry to demonstrate efficient electron transfer. The nanomaterial shows high performance in the electrochemical detection of DNA hybridization leading to a variation in the electrochemical signal of ferrocene with a detection limit of 0.3 fM. Furthermore, the biosensor demonstrates ability for sensing DNA of rpoB gene of Mycobacterium tuberculosis in real PCR samples. Developed biosensor was suitable for detection of sequences with a single nucleotide polymorphism (SNP) T (TCG/TTG), responsible for resistance of M. tuberculosis to rifampicin drug, and discriminating them from wild-type samples without such mutation. This shows potential of such systems for further application in pathogens diagnostic and therapeutic purpose.

Plasmonic ruler on field-effect devices for kinase drug discovery applications.

Protein kinases are cellular switches that mediate phosphorylation of proteins. Abnormal phosphorylation of proteins is associated with lethal diseases such as cancer. In the pharmaceutical industry, protein kinases have become an important class of drug targets. This study reports a versatile approach for the detection of protein phosphorylation. The change in charge of the myelin basic protein upon phosphorylation by the protein kinase C-alpha (PKC-α) in the presence of adenosine 5'-[γ-thio] triphosphate (ATP-S) was detected on gold metal-insulator-semiconductor (Au-MIS) capacitor structures. Gold nanoparticles (AuNPs) can then be attached to the thio-phosphorylated proteins, forming a Au-film/AuNP plasmonic couple. This was detected by a localized surface plasmon resonance (LSPR) technique alongside MIS capacitance. All reactions were validated using surface plasmon resonance technique and the interaction of AuNPs with the thio-phosphorylated proteins quantified by quartz crystal microbalance. The plasmonic coupling was also visualized by simulations using finite element analysis. The use of this approach in drug discovery applications was demonstrated by evaluating the response in the presence of a known inhibitor of PKC-α kinase. LSPR and MIS on a single platform act as a cross check mechanism for validating kinase activity and make the system robust to test novel inhibitors.

Electrochemical detection of the oligomerization of PB1-F2 influenza A virus protein in infected cells.

PB1-F2 is a nonstructural accessory protein of Influenza A virus described to enhance the mortality and the morbidity of the virus in a host-dependent manner. In this work, an electrochemical biosensor based on an immunodetection system was developed to follow the oligomerization of PB1-F2 during the viral cycle. The immunosensor was based on conductive polypyrrole modified with ferrocenyl groups as a redox marker for enhancing signal detection. Antibodies specific for monomeric or oligomeric PB1-F2 forms were immobilized on polypyrrole matrix via biotin/streptavidin layer. We demonstrated that this electrochemical biosensor sensitively detects PB1-F2 in both conformational forms. The linear range extends from 5 nM to 1.5 µM and from 5 nM to 0.5 µM for monomeric and oligomeric PB1-F2, respectively. The calculated limit of detection was 0.42 nM for monomeric PB1-F2 and 16 nM for oligomers. The biosensor platform allows the detection and quantification of PB1-F2 in lysates of infected cells during viral cycle. We show that at early stages of viral cycle, PB1-F2 is mainly monomeric but switched to amyloid-like structures at a later stage of infection. The quantification of two protein structural forms points out that PB1-F2 expression profiles and kinetics of oligomerization are cell-type-dependent.

Direct electrochemical detection of PB1-F2 protein of influenza A virus in infected cells.

Influenza virus represents a major concern of human health and animal production. PB1-F2 is a small proapoptotic protein supposed to contribute to the virulence of influenza A virus (IAV). However, the molecular mechanism of action of PB1-F2 is still unclear.PB1-F2 expression and behavior during the viral cycle is difficult to follow with classical biochemical methods. In this work we have developed an electrochemical biosensor based on immuno-detection system for quantification of PB1-F2 protein in infected cell. The electrochemical immunosensor was based on conducting copolypyrrole integrating ferrocenyl group as redox marker for enhancing signal detection. A specific anti-PB1-F2 monoclonal antibody was immobilized on the copolypyrrole layer via biotin-streptavidin system. We demonstrate that this electrochemical system sensitively detect purified recombinant PB1-F2 over a wide range of concentrations from 5 nM to 1.5 µM. The high sensor sensitivity allowed the detection of PB1-F2 in lysates of infected cells confirming that PB1-F2 is expressed in early stages of viral cycle. The immunosensor developed shows enhanced performances for the evaluation of PB1-F2 protein concentration in biological samples and could be applied for studying of PB1-F2 during influenza virus infection.

Electrochemical aptasensor of human cellular prion based on multiwalled carbon nanotubes modified with dendrimers: a platform for connecting redox markers and aptamers.

The present work aims to develop an electrochemical biosensor based on aptamer able to detect human cellular prions PrP(C) as a model biomarker of prion disease with high sensitivity. We designed the biosensor using multiwalled carbon nanotubes (MWCNTs) modified with polyamidoamine dendrimers of fourth generation (PAMAM G4) which in turn were coupled to DNA aptamers used as bioreceptors. Electrochemical signal was detected by a ferrocenyl redox marker incorporated between the dendrimers and aptamers interlayer. MWCNTs, thanks to their nanostructure organization and electrical properties, allow the distribution of aptamers and redox markers over the electrode surface. We demonstrated that the interaction between aptamers and prion proteins leads to variation in the electrochemical signal of the ferrocenyl group. High sensitivity with a detection limit of 0.5 pM and a wide linear range of detection from 1 pM to 10 µM has been demonstrated. Detection of PrP(C) in spiked blood plasma has been achieved in the same range of concentrations as for detection of PrP(C) in buffer. The sensor demonstrated a recovery of minimum 85% corresponding to 1 nM PrP(C) and a maximum of 127% corresponding to 1 pM PrP(C).

Characterization of structure and activity of garlic peroxidase (POX(1B)).

Structural characterization and study of the activity of new POX(1B) protein from garlic which has a high peroxidase activity and can be used as a biosensor for the detection of hydrogen peroxide and phenolic compounds were performed and compared with the findings for other heme peroxidases. The structure-function relationship was investigated by analysis of the spectroscopic properties and correlated to the structure determined by a new generation of high-performance hybrid mass spectrometers. The reactivity of the enzyme was analyzed by studies of the redox activity toward various ligands and the reactivity with various substrates. We demonstrated that, in the case of garlic peroxidase, the heme group is pentacoordinated, and has an histidine as a proximal ligand. POX(1B) exhibited a high affinity for hydrogen peroxide as well as various reducing cosubstrates. In addition, high enzyme specificity was demonstrated. The k(cat) and K(M) values were 411 and 400 mM(-1) s(-1) for 3,3',5,5'-tetramethylbenzidine and 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), respectively. Furthermore, the reduction of nitro compounds in the presence of POX(1B) was demonstrated by iron(II) nitrosoalkane complex assay. In addition, POX(1B) showed a great potential for application for drug metabolism since its ability to react with 1-nitrohexane in the presence of sodium dithionite was demonstrated by the appearance of a characteristic Soret band at 411 nm. The high catalytic efficiency obtained in the case of the new garlic peroxidase (POX(1B)) is suitable for the monitoring of different analytes and biocatalysis.

[Masked depression--history and present days]. / Depresja maskowana--historia i wspólczesnosc.

Masked depression is a special form of an atypical depression. In the 70's and 80's years it was often identified at patients who complained on somatic diseases, without any distinguishable organic disorder. Depression symptoms were of secondary importance, with lesser intensification, some of them didn't appear at all. The psychiatrists of the time created a lot of terms to describe them, i.e.: a depression equivalent, a vegetative equivalent, a depression without a depression, a hidden depression. Current classifications: ICD-10 (International Classification of Diseases, 10th Revision) and DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, published by the American Psychiatric Association) do not contain the term masked depression. It doesn't mean that have disappeared the problem of atypical depression syndrome with a picture significantly different from the commonly known. The american scientists claim that such group contains 6-7% of depression disorders. The lack of proper diagnosis and disease entity qualification leads to serious somatic and psychological consequences for patients. Improper diagnosis and treatment of a patient limits his or her social and professional life, forms suicide rate and makes high costs of diagnosis and treatment.