Detection of α-Galactosidase A Reaction in Samples Extracted from Dried Blood Spots Using Ion-Sensitive Field Effect Transistors.

Fabry disease is a lysosomal storage disorder caused by a significant decrease in the activity or absence of the enzyme α-galactosidase A. The diagnostics of Fabry disease during newborn screening are reasonable, due to the availability of enzyme replacement therapy. This paper presents an electrochemical method using complementary metal-oxide semiconductor (CMOS)-compatible ion-sensitive field effect transistors (ISFETs) with hafnium oxide-sensitive surfaces for the detection of α-galactosidase A activity in dried blood spot extracts. The capability of ISFETs to detect the reaction catalyzed by α-galactosidase A was demonstrated. The buffer composition was optimized to provide suitable conditions for both enzyme and ISFET performance. The use of ISFET structures as sensor elements allowed for the label-free detection of enzymatic reactions with melibiose, a natural substrate of α-galactosidase A, instead of a synthetic fluorogenic one. ISFET chips were packaged with printed circuit boards and microfluidic reaction chambers to enable long-term signal measurement using a custom device. The packaged sensors were demonstrated to discriminate between normal and inhibited GLA activity in dried blood spots extracts. The described method offers a promising solution for increasing the widespread distribution of newborn screening of Fabry disease.

A Portable Readout System for Biomarker Detection with Aptamer-Modified CMOS ISFET Array.

Biosensors based on ion-sensitive field effect transistors (ISFETs) combined with aptamers offer a promising and convenient solution for point-of-care testing applications due to the ability for fast and label-free detection of a wide range of biomarkers. Mobile and easy-to-use readout devices for the ISFET aptasensors would contribute to further development of the field. In this paper, the development of a portable PC-controlled device for detecting aptamer-target interactions using ISFETs is described. The device assembly allows selective modification of individual ISFETs with different oligonucleotides. Ta2O5-gated ISFET structures were optimized to minimize trapped charge and capacitive attenuation. Integrated CMOS readout circuits with linear transfer function were used to minimize the distortion of the original ISFET signal. An external analog signal digitizer with constant voltage and superimposed high-frequency sine wave reference voltage capabilities was designed to increase sensitivity when reading ISFET signals. The device performance was demonstrated with the aptamer-driven detection of troponin I in both reference voltage setting modes. The sine wave reference voltage measurement method reduced the level of drift over time and enabled a lowering of the minimum detectable analyte concentration. In this mode (constant voltage 2.4 V and 10 kHz 0.1Vp-p), the device allowed the detection of troponin I with a limit of detection of 3.27 ng/mL. Discrimination of acute myocardial infarction was demonstrated with the developed device. The ISFET device provides a platform for the multiplexed detection of different biomarkers in point-of-care testing.

Off-Stoichiometry Thiol-Ene Polymers: Inclusion of Anchor Groups Using Allylsilanes.

The use of polymers in silicon chips is of great importance for the development of microelectronic and biomedical industries. In this study, new silane-containing polymers, called OSTE-AS polymers, were developed based on off-stoichiometry thiol-ene polymers. These polymers can bond to silicon wafers without pretreatment of the surface by an adhesive. Silane groups were included in the polymer using allylsilanes, with the thiol monomer as the target of modification. The polymer composition was optimized to provide the maximum hardness, the maximum tensile strength, and good bonding with the silicon wafers. The Young's modulus, wettability, dielectric constant, optical transparency, TGA and DSC curves, and the chemical resistance of the optimized OSTE-AS polymer were studied. Thin OSTE-AS polymer layers were obtained on silicon wafers via centrifugation. The possibility of creating microfluidic systems based on OSTE-AS polymers and silicon wafers was demonstrated.

Regulatory miPEP Open Reading Frames Contained in the Primary Transcripts of microRNAs.

This review aims to consider retrospectively the available data on the coding properties of pri-microRNAs and the regulatory functions of their open reading frames (ORFs) and the encoded peptides (miPEPs). Studies identifying miPEPs and analyzing the fine molecular mechanisms of their functional activities are reviewed together with a brief description of the methods to identify pri-miRNA ORFs and the encoded protein products. Generally, miPEPs have been identified in many plant species of several families and in a few animal species. Importantly, molecular mechanisms of the miPEP action are often quite different between flowering plants and metazoan species. Requirement for the additional studies in these directions is highlighted by alternative findings concerning negative or positive regulation of pri-miRNA/miRNA expression by miPEPs in plants and animals. Additionally, the question of how miPEPs are distributed in non-flowering plant taxa is very important for understanding the evolutionary origin of such micropeptides. Evidently, further extensive studies are needed to explore the functions of miPEPs and the corresponding ORFs and to understand the full set of their roles in eukaryotic organisms. Thus, we address the most recent integrative views of different genomic, physiological, and molecular aspects concerning the expression of miPEPs and their possible fine functions.

Minibody-Based and scFv-Based Antibody Fragment-Drug Conjugates Selectively Eliminate GD2-Positive Tumor Cells.

Ganglioside GD2 is a well-established target expressed on multiple solid tumors, many of which are characterized by low treatment efficiency. Antibody-drug conjugates (ADCs) have demonstrated marked success in a number of solid tumors, and GD2-directed drug conjugates may also hold strong therapeutic potential. In a recent study, we showed that ADCs based on the approved antibody dinutuximab and the drugs monomethyl auristatin E (MMAE) or F (MMAF) manifested potent and selective cytotoxicity in a panel of tumor cell lines and strongly inhibited solid tumor growth in GD2-positive mouse cancer models. Here, we employed two different GD2-binding moieties-minibodies and scFv fragments that carry variable antibody domains identical to those of dinutuximab, and site-directly conjugated them to MMAE or MMAF by thiol-maleimide chemistry with drug-to-antibody ratios (DAR) of 2 and 1, respectively. Specific binding of the antibody fragment-drug conjugates (FDCs) to GD2 was confirmed in direct ELISA, flow cytometry, and confocal microscopy. Selective cytotoxic and cytostatic effects of the conjugates were observed in GD2-positive but not GD2-negative neuroblastoma and melanoma cell lines. Minibody-based FDCs demonstrated more pronounced cytotoxic effects and stronger antigen binding compared to scFv-based FDCs. The developed molecules may offer considerable practical benefit, since antibody fragment-drug conjugates are capable of enhancing therapeutic efficacy of ADCs by improving their pharmacokinetic characteristics and reducing side effects.

Off-Stoichiometry Thiol-Enes Polymers Containing Silane Groups for Advanced Packaging Technologies.

New modified off-stoichiometry thiol-enes polymers, called OSTE-MS polymers, were developed by introducing mercaptosilane into the polymer mixture. This modification made it possible to introduce silane groups into the polymer frame, due to which the polymer gained the ability to bond with silicon wafers without modification of the wafer surface by any adhesive. The optimal composition for creating 3D polymer structures on a chip was selected, which consists of a volume ratio of 6:6:1 of allyl monomer, mercapto monomer, and mercaptosilane, respectively. The hardness, shift force, tensile strength, Young's modulus, optical transparency, glass transition temperature, thermal stability, and chemical resistance of the OSTE-MS polymer, and the viscosity for the prepolymer mixture were studied. On the basis of the OSTE-MS polymer, 3D polymer structures of the well type and microfluidic system on the silicon chips were obtained.

7,8-Dihydro-8-oxo-1,N6-ethenoadenine: an exclusively Hoogsteen-paired thymine mimic in DNA that induces A→T transversions in Escherichia coli.

This work investigated the structural and biological properties of DNA containing 7,8-dihydro-8-oxo-1,N6-ethenoadenine (oxo-ϵA), a non-natural synthetic base that combines structural features of two naturally occurring DNA lesions (7,8-dihydro-8-oxoadenine and 1,N6-ethenoadenine). UV-, CD-, NMR spectroscopies and molecular modeling of DNA duplexes revealed that oxo-ϵA adopts the non-canonical syn conformation (χ = 65º) and fits very well among surrounding residues without inducing major distortions in local helical architecture. The adduct remarkably mimics the natural base thymine. When considered as an adenine-derived DNA lesion, oxo-ϵA was >99% mutagenic in living cells, causing predominantly A→T transversion mutations in Escherichia coli. The adduct in a single-stranded vector was not repaired by base excision repair enzymes (MutM and MutY glycosylases) or the AlkB dioxygenase and did not detectably affect the efficacy of DNA replication in vivo. When the biological and structural data are viewed together, it is likely that the nearly exclusive syn conformation and thymine mimicry of oxo-ϵA defines the selectivity of base pairing in vitro and in vivo, resulting in lesion pairing with A during replication. The base pairing properties of oxo-ϵA, its strong fluorescence and its invisibility to enzymatic repair systems in vivo are features that are sought in novel DNA-based probes and modulators of gene expression.

Probing GFP Chromophore Analogs as Anti-HIV Agents Targeting LTR-III G-Quadruplex.

Green fluorescent protein (GFP) chromophore and its congeners draw significant attention mostly for bioimaging purposes. In this work we probed these compounds as antiviral agents. We have chosen LTR-III DNA G4, the major G-quadruplex (G4) present in the long terminal repeat (LTR) promoter region of the human immunodeficiency virus-1 (HIV-1), as the target for primary screening and designing antiviral drug candidates. The stabilization of this G4 was previously shown to suppress viral gene expression and replication. FRET-based high-throughput screening (HTS) of 449 GFP chromophore-like compounds revealed a number of hits, sharing some general structural features. Structure-activity relationships (SAR) for the most effective stabilizers allowed us to establish structural fragments, important for G4 binding. Synthetic compounds, developed on the basis of SAR analysis, exhibited high LTR-III G4 stabilization level. NMR spectroscopy and molecular modeling revealed the possible formation of LTR-III G4-ligand complex with one of the lead selective derivative ZS260.1 positioned within the cavity, thus supporting the LTR-III G4 attractiveness for drug targeting. Selected compounds showed moderate activity against HIV-I (EC50 1.78-7.7 µM) in vitro, but the activity was accompanied by pronounced cytotoxicity.

Extracellular heat shock proteins and cancer: New perspectives.

Heat shock proteins (HSPs) are a large family of molecular chaperones aberrantly expressed in cancer. The expression of HSPs in tumor cells has been shown to be implicated in the regulation of apoptosis, immune responses, angiogenesis and metastasis. Given that extracellular vesicles (EVs) can serve as potential source for the discovery of clinically useful biomarkers and therapeutic targets, it is of particular interest to study proteomic profiling of HSPs in EVs derived from various biological fluids of cancer patients. Furthermore, a divergent expression of circulating microRNAs (miRNAs) in patient samples has opened new opportunities in exploiting miRNAs as diagnostic tools. Herein, we address the current literature on the expression of extracellular HSPs with particular interest in HSPs in EVs derived from various biological fluids of cancer patients and different types of immune cells as promising targets for identification of clinical biomarkers of cancer. We also discuss the emerging role of miRNAs in HSP regulation for the discovery of blood-based biomarkers of cancer. We outline the importance of understanding relationships between various HSP networks and co-chaperones and propose the model for identification of HSP signatures in cancer. Elucidating the role of HSPs in EVs from the proteomic and miRNAs perspectives may provide new opportunities for the discovery of novel biomarkers of cancer.

Integration of a field effect transistor-based aptasensor under a hydrophobic membrane for bioelectronic nose applications.

A new bioelectronic nose based on a field effect transistor coupled with an aptamer as the sensing element was developed. The gas-to-liquid extraction interface required for appropriate aptamer function was integrated into standard CMOS technology. It was developed with the use of a sacrificial aluminium etching technique combined with surface modifications by silanes for wettability control. As a proof of concept, aptamer Van74 for vanillin was immobilized on the sensitive surface of the ISFET. The developed microsystem can selectively detect vanillin vapor in a concentration range from 2.7 ppt to 0.3 ppm, with a detection limit of 2.7 ppt. The sensor was able to detect vanillin in a gas sample obtained from roasted coffee beans. This outcome provides a foundation for developing a new generation of bioelectronic noses for the detection and discrimination of volatile compounds.

Development of the covalent antibody-DNA conjugates technology for detection of IgE and IgM antibodies by immuno-PCR.

Immuno-PCR (iPCR) is one of the methods used for the detection of a wide range of analytes and features the high sensitivity of the polymerase chain reaction (PCR) method. iPCR uses antibodies coupled to DNA, followed by the amplification of the attached DNA using RT-PCR. Two major types of antibody-DNA conjugates are currently used, which are obtained as a result of non-covalent (biotin-streptavidin) or covalent interactions. Using a strain-promoted azide-alkyne cycloaddition (SPAAC), we synthesized covalent DNA-antibody conjugates, optimized the reaction conditions, and developed an efficient protocol for the purification of conjugates, with which all unreacted antibodies and oligonucleotides are separated. Covalent DNA-antibody conjugates were tested with iPCR assays that were previously developed for the detection of IgE and IgM antibodies with the use of the supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin. The results show that the modification of antibodies with amino groups did not allow us to obtain monolabeled antibodies or antibodies with a strictly defined number of DNA-labels. The degree of labeling determined by the dyes introduced through the azido group reflects the actual labeling degree statistically. If the average labeling degree for azido groups is 1.1, the conjugates contain 25% mono-labeled antibodies, 50% double-labeled antibodies, and 25% unlabeled ones. The specificity of the monoclonal antibody to human IgE (BE5) changed after conjugation with the oligonucleotide. The sensitivity of iPCR in the detection of IgM antibodies produced against the LeC disaccharide using a covalent conjugate was similar to that of a supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin, but the new procedure is two steps shorter.

Immuno-PCR technology for detection of natural human antibodies against Lec disaccharide.

The development of an immuno-PCR assay for quantitation of low amounts of anti-glycan human antibodies is described. The sensitivity of the assay for determination of low-affinity anti-LeC IgM has been found to be 4 ng/ml (~100 pg per sample), thus being two orders of magnitude higher compared to the conventional ELISA with the same antigen.