Aptamers vs. antibodies as capture probes in optical porous silicon biosensors.

Over the past decade aptamers have emerged as a promising class of bioreceptors for biosensing applications with significant advantages over conventional antibodies. However, experimental studies comparing aptasensors and immunosensors, under equivalent conditions, are limited and the results are inconclusive, in terms of benefits and limitations of each bioreceptor type. In the present work, the performance of aptamer and antibody bioreceptors for the detection of a his-tagged protein, used as a model target, is compared. The bioreceptors are immobilized onto a nanostructured porous silicon (PSi) thin film, used as the optical transducer, and the target protein is detected in a real-time and label-free format by reflective interferometric Fourier transform spectroscopy. For the antibodies, random-oriented immobilization onto the PSi nanostructure results in a poor biosensing performance. Contrary, Fc-oriented immobilization of the antibodies shows a similar biosensing performance to that exhibited by the aptamer-based biosensor, in terms of binding rate, dynamic detection range, limit of detection and selectivity. The aptasensor outperforms in terms of its reusability and storability, while the immunosensor could not be regenerated for subsequent experiments.

All-Optical Planar Polymer Waveguide-Based Biosensor Chip Designed for Smartphone-Assisted Detection of Vitamin D.

An all-optical plasmonic sensor platform designed for smartphones based on planar-optical waveguide structures integrated in a polymer chip is reported for the first time. To demonstrate the applicability of the sensor system for biosensing purposes, the detection of 25-hydroxyvitamin D (25OHD) in human serum samples using an AuNP-enhanced aptamer-based assay was demonstrated. With the aid of the developed assay sensitivity of 0.752 pixel/nM was achieved for 25OHD concentrations ranging from 0-100 nM. The waveguide structure of the sensor enables miniaturisation and parallelisation, thus, demonstrates the potential for simultaneous detection of various analytes including biomarkers. The entire optical arrangement can be integrated into a single polymer chip which allows for large scale and cost-efficient sensor fabrication. The broad utilization and access of smartphone electronics make the proposed design most attractive for its wider use in lab-on-chip applications.

SPR Biosensor Based on Polymer Multi-Mode Optical Waveguide and Nanoparticle Signal Enhancement.

We present a surface plasmon resonance (SPR) biosensor that is based on a planar-optical multi-mode (MM) polymer waveguide structure applied for the detection of biomolecules in the lower nano-molar (nM) range. The basic sensor shows a sensitivity of 608.6 nm/RIU when exposed to refractive index changes with a measurement resolution of 4.3 × 10-3 RIU. By combining the SPR sensor with an aptamer-functionalized, gold-nanoparticle (AuNP)-enhanced sandwich assay, the detection of C-reactive protein (CRP) in a buffer solution was achieved with a response of 0.118 nm/nM. Due to the multi-mode polymer waveguide structure and the simple concept, the reported biosensor is well suited for low-cost disposable lab-on-a-chip applications and can be used with rather simple and economic devices. In particular, the sensor offers the potential for fast and multiplexed detection of several biomarkers on a single integrated platform.

Aptamer-Modified Magnetic Beads in Biosensing.

Magnetic beads (MBs) are versatile tools for the purification, detection, and quantitative analysis of analytes from complex matrices. The superparamagnetic property of magnetic beads qualifies them for various analytical applications. To provide specificity, MBs can be decorated with ligands like aptamers, antibodies and peptides. In this context, aptamers are emerging as particular promising ligands due to a number of advantages. Most importantly, the chemical synthesis of aptamers enables straightforward and controlled chemical modification with linker molecules and dyes. Moreover, aptamers facilitate novel sensing strategies based on their oligonucleotide nature that cannot be realized with conventional peptide-based ligands. Due to these benefits, the combination of aptamers and MBs was already used in various analytical applications which are summarized in this article.

Aqueous Synthesis of PEGylated Quantum Dots with Increased Colloidal Stability and Reduced Cytotoxicity.

Ligands used on the surface of colloidal nanoparticles (NPs) have a significant impact on physiochemical properties of NPs and their interaction in biological environments. In this study, we report a one-pot aqueous synthesis of 3-mercaptopropionic acid (MPA)-functionalized CdTe/CdS/ZnS quantum dots (Qdots) in the presence of thiol-terminated methoxy polyethylene glycol (mPEG) molecules as a surface coordinating ligand. The resulting mPEG-Qdots were characterized by using ζ potential, FTIR, thermogravimetric (TG) analysis, and microscale thermophoresis (MST) studies. We investigated the effect of mPEG molecules and their grafting density on the Qdots photophysical properties, colloidal stability, protein binding affinity, and in vitro cellular toxicity. Moreover, cellular binding features of the resulting Qdots were examined by using three-dimensional (3D) tumor-like spheroids, and the results were discussed in detail. Promisingly, mPEG ligands were found to increase colloidal stability of Qdots, reduce adsorption of proteins to the Qdot surface, and mitigate Qdot-induced side effects to a great extent. Flow cytometry and confocal microscopy studies revealed that PEGylated Qdots exhibited distinctive cellular interactions with respect to their mPEG grafting density. As a result, mPEG molecules demonstrated a minimal effect on the ZnS shell deposition and the Qdot fluorescence efficiency at a low mPEG density, whereas they showed pronounced effect on Qdot colloidal stability, protein binding affinity, cytotoxicity, and nonspecific binding at a higher mPEG grafting amount.

Label-free optical biosensors based on aptamer-functionalized porous silicon scaffolds.

A proof-of-concept for a label-free and reagentless optical biosensing platform based on nanostructured porous silicon (PSi) and aptamers is presented in this work. Aptamers are oligonucleotides (single-stranded DNA or RNA) that can bind their targets with high affinity and specificity, making them excellent recognition elements for biosensor design. Here we describe the fabrication and characterization of aptamer-conjugated PSi biosensors, where a previously characterized his-tag binding aptamer (6H7) is used as model system. Exposure of the aptamer-functionalized PSi to the target proteins as well as to complex fluids (i.e., bacteria lysates containing target proteins) results in robust and well-defined changes in the PSi optical interference spectrum, ascribed to specific aptamer-protein binding events occurring within the nanoscale pores, monitored in real time. The biosensors show exceptional stability and can be easily regenerated by a short rinsing step for multiple biosensing analyses. This proof-of-concept study demonstrates the possibility of designing highly stable and specific label-free optical PSi biosensors, employing aptamers as capture probes, holding immense potential for application in detection of a broad range of targets, in a simple yet reliable manner.

Identification of the target binding site of ethanolamine-binding aptamers and its exploitation for ethanolamine detection.

Aptamers are promising recognition elements for sensitive and specific detection of small molecules. We have previously selected ssDNA aptamers for ethanolamine, one of the smallest aptamer targets so far. The work presented here focuses on the determination of the binding region within the aptamer structure and its exploitation for the development of an aptamer-based assay for detection of ethanolamine. Sequence analysis of the aptamers resulted in the identification of a G-rich consensus sequence, which was able to fold in a typical two- or three-layered G-quartet structure. Experiments with stepwise truncated variants of the aptamers revealed that the consensus sequence is responsible and sufficient for binding to the target. On the basis of the knowledge of the aptamers binding site, we developed an aptamer-based microarray assay relying on competition between ethanolamine and an oligonucleotide complementary to the consensus sequence. Competitive binding of ethanolamine and fluorescently labeled complementary oligonucleotides resulted in fluorescence intensities dependent on ethanolamine concentration with a limit of detection of 10 pM. This method enables detection of small molecules without any labeling of analytes. The competitive assay could potentially be transferred to other aptamers and thus provides a promising system for aptamer-based detection of diverse small molecules.

New, non-quinone fluorogeldanamycin derivatives strongly inhibit Hsp90.

Streptomyces hygroscopicus is a natural producer of geldanamycin. Mutasynthetic supplementation of an AHBA-blocked mutant with all possible monofluoro 3-aminobenzoic acids provided new fluorogeldanamycins. These showed strong antiproliferative activity and inhibitory effects on human heat shock protein Hsp90. Binding to Hsp90 in the low nanomolar range was determined from molecular modelling, AFM analysis and by calorimetric studies.

Analysis of oxygen-dependent cytokine expression in human mesenchymal stem cells derived from umbilical cord.

Efficient cell expansion is a basic requirement for obtaining clinically relevant numbers of mesenchymal stem cells designed for cell-based therapies or tissue-engineering application. Previous studies have demonstrated that mesenchymal stem cells (MSC) cultivated under reduced atmospheric oxygen concentrations (2.5% O2) possess enhanced proliferation potential and can maintain their differentiation properties. We have analyzed the oxygen-dependent cytokine expression of human MSC derived from umbilical cord and attempted to link the results to the proliferation and differentiation capacities of these cells. By quantitative reverse transcription plus the polymerase chain reaction and by protein microarray, we measured the gene expression and intracellular protein concentration of several growth factors and growth factor receptors. Fibroblast growth factor-7, two growth factor receptors (vascular endothelial growth factor receptor 2 and stem cell factor receptor), and two growth-factor-binding proteins (insulin-like growth-factor-binding proteins 3 and 6) were over-expressed under hypoxic conditions, indicating that their signaling pathways participate in cell proliferation. On the other hand, typical differentiation factors such as bone morphogenetic protein-4, endothelial growth factor, and tissue growth factor-ß1 were absent in cells cultivated under hypoxic and normoxic conditions. The absolute concentration of some intracellular cytokines was also measured for the first time under hypoxia and normoxia. Our results in combination with previous findings indicate that enhanced proliferation potential and a maintained undifferentiated cell state can be ascribed to the oxygen-dependent expression of a set of cytokines. This knowledge might help in the understanding of MSC physiology and in the achievement of directed cell fate of MSC for clinical application.

Aptamers: versatile probes for flow cytometry.

Aptamers are nucleic acid oligomers with distinct conformational shapes that allow them to bind targets with high affinity and specificity. Aptamers are selected from a random oligonucleotide library by their capability to bind a certain molecular target. A variety of targets ranging from small molecules like amino acids to complex targets and whole cells have been used to select aptamers. These characteristics and the ability to create specific aptamers against virtually any cell type in a process termed "systematic evolution by exponential enrichment" make them interesting tools for flow cytometry. In this contribution, we review the application of aptamers as probes for flow cytometry, especially cell-phenotyping and detection of various cancer cell lines and virus-infected cells and pathogens. We also discuss the potential of aptamers combined with nanoparticles such as quantum dots for the generation of new multivalent detector molecules with enhanced affinity and sensitivity. With regard to recent advancements in aptamer selection and the decreasing costs for oligonucleotide synthesis, aptamers may rise as potent competitors for antibodies as molecular probes in flow cytometry.

Identification of Major Constituents of Hypericum perforatum L. Extracts in Syria by Development of a Rapid, Simple, and Reproducible HPLC-ESI-Q-TOF MS Analysis and Their Antioxidant Activities.

Hypericum perforatum Linn (St. John's wort) is a popular and widespread medicine in Syria, which is used for a wide range of conditions, including gastrointestinal diseases, heart disease, skin diseases, and psychological disorders. This widespread use prompted us to identify the main compounds of this plant from Syria that are responsible for its medicinal properties, especially since its components differ between countries according to the nature of the soil, climate, and altitude. This is, to the best of our knowledge, the first report in which St. John's wort, a plant native to Syria, is extracted using different solvents and its most important compounds are identified. In this study, the dried above-ground parts, i.e., leaves, stem, petals, and flowers, were extracted using different solvents (water, ethanol, methanol, and acetone) and extraction protocols. By increasing the polarity of the solvent, higher yields were obtained, indicating that mainly hydrophobic compounds were extracted. Therefore, we conclude that extraction using the tea method or using a mixture of water and organic solvents resulted in higher yields compared with pure organic solvents or continuous boiling with water for long periods. The obtained extracts were analyzed using high-performance liquid chromatography equipped with a diode array detector (HPLC-DAD), coupled with UV-visible spectrophotometry at a full spectrum (200-800 nm). The HPLC spectra of the extracts were almost identical at three wavelengths (260 nm for phloroglucinols (hyperforin and derivates), 590 nm for naphthodianthrones (hypericins), and 350 nm for other flavonols, flavones, and caffeoylquinic acids), with differences observed only in the intensity of the peaks. This indicates that the same compounds were obtained using different solvents, but in different amounts. Five standards (chlorogenic acid, quercetin, quercitrin hydrate, hyperoside, and hypericin) were used, and a comparison with retention times and ultraviolet (UV) spectra reported in the literature was performed to identify 10 compounds in these extracts: hyperforin, adhyperforin, hypericin, rutin, quercetin, quercitrin, quercitrin hydrate, hyperoside, biapigenin, and chlorogenic acid. Although the European Pharmacopoeia still describes ultraviolet spectroscopy as a method for determining the quantity of Hyperici herba, interference from other metabolites can occur. Combined HPLC-DAD and electrospray ionization-mass spectrometry (LC-ESI-MS) in the positive mode have therefore also been used to confirm the presence of these compounds in the extracts by correlating known masses with the identified masses or through characteristic fragmentation patterns. Total phenolic contents of the extracts were determined by the Folin-Ciocalteu assay, and antioxidant activity was evaluated as free radical scavenging capacity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. The results indicate that the aqueous extracts prepared by the tea method gave the highest total phenols, while the pure organic solvents gave very low phenols. Also, the extracts that contain the largest amount of phenols gave lower IC50 values or higher antioxidant activity than that of others.

Green Synthesis of Silver Nanoparticles Using Hypericum perforatum L. Aqueous Extract with the Evaluation of Its Antibacterial Activity against Clinical and Food Pathogens.

The rapid development of nanotechnology and its applications in medicine has provided the perfect solution against a wide range of different microbes, especially antibiotic-resistant ones. In this study, a one-step approach was used in preparing silver nanoparticles (AgNPs) by mixing silver nitrate with hot Hypericum perforatum (St. John's wort) aqueous extract under high stirring to prevent agglomeration. The formation of silver nanoparticles was monitored by continuous measurement of the surface plasma resonance spectra (UV-VIS). The effect of St. John's wort aqueous extract on the formation of silver nanoparticles was evaluated and fully characterized by using different physicochemical techniques. The obtained silver nanoparticles were spherical, monodisperse, face-centered cubic (fcc) crystal structures, and the size ranges between 20 to 40 nm. They were covered with a capping layer of organic compounds considered as a nano dimension protective layer that prevents agglomeration and sedimentation. AgNPs revealed antibacterial activity against both tested Gram-positive and Gram-negative bacterial strains causing the formation of 13-32 mm inhibition zones with MIC 6.25-12.5 µg/mL; Escherichia coli strains were resistant to tested AgNPs. The specific growth rate of S. aureus was significantly reduced due to tested AgNPs at concentrations ≥½ MIC. AgNPs did not affect wound migration in fibroblast cell lines compared to control. Our results highlighted the potential use of AgNPs capped with plant extracts in the pharmaceutical and food industries to control bacterial pathogens' growth; however, further studies are required to confirm their wound healing capability and their health impact must be critically evaluated.

Aptamer-based downstream processing of his-tagged proteins utilizing magnetic beads.

Aptamers are synthetic nucleic acid-based high affinity ligands that are able to capture their corresponding target via molecular recognition. Here, aptamer-based affinity purification for His-tagged proteins was developed. Two different aptamers directed against the His-tag were immobilized on magnetic beads covalently. The resulting aptamer-modified magnetic beads were characterized and successfully applied for purification of different His-tagged proteins from complex E. coli cell lysates. Purification effects comparable to conventional immobilized metal affinity chromatography were achieved in one single purification step. Moreover, we have investigated the possibility to regenerate and reuse the aptamer-modified magnetic beads and have shown their long-term stability over a period of 6 months.

Aptamer-Modified Hydrogels.

Hydrogels have attracted much attention especially due to their biocompatibility and their potential for stimulus responsiveness. By combining hydrogels with aptamers, biological recognition and responsiveness can be added to hydrogels, thereby opening path to advanced applications in biosensing and biomedicine. Within this chapter aptamers will be introduced and their contributions to biological responsiveness of hydrogels will be described. Especially the aptamer-based mechanisms that result in biological responsiveness will be explained and examples for the application of these mechanisms will be given ranging from rather simple sensing approaches to advanced materials for tissue engineering and drug delivery. Since aptamers are not only highly specific bioreceptors, but represent switchable structures that can be easily manipulated using well-known DNA techniques, the combination of aptamers and hydrogels facilitates the rational design of well-programmable and target-responsive smart hydrogels.

Hypericum perforatum L.-Mediated Green Synthesis of Silver Nanoparticles Exhibiting Antioxidant and Anticancer Activities.

This contribution focuses on the green synthesis of silver nanoparticles (AgNPs) with a size < 100 nm for potential medical applications by using silver nitrate solution and Hypericum Perforatum L. (St John's wort) aqueous extracts. Various synthesis methods were used and compared with regard to their yield and quality of obtained AgNPs. Monodisperse spherical nanoparticles were generated with a size of approximately 20 to 50 nm as elucidated by different techniques (SEM, TEM). XRD measurements showed that metallic silver was formed and the particles possess a face-centered cubic structure (fcc). SEM images and FTIR spectra revealed that the AgNPs are covered by a protective surface layer composed of organic components originating from the plant extract. Ultraviolet-visible spectroscopy, dynamic light scattering, and zeta potential were also measured for biologically synthesized AgNPs. A potential mechanism of reducing silver ions to silver metal and protecting it in the nanoscale form has been proposed based on the obtained results. Moreover, the AgNPs prepared in the present study have been shown to exhibit a high antioxidant activity for 2, 2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation, and super oxide anion radical and 2,2-diphenyl-1-picrylhydrazyl. Synthesized AgNPs showed high cytotoxicity by inhibiting cell viability for Hela, Hep G2, and A549 cells.

Aptazymes: Expanding the Specificity of Natural Catalytic Nucleic Acids by Application of In Vitro Selected Oligonucleotides.

Aptazymes are synthetic molecules composed of an aptamer domain and a catalytic active nucleic acid unit, which may be a ribozyme or a DNAzyme. In these constructs the aptamer domain serves as a molecular switch that can regulate the catalytic activity of the ribozyme or DNAzyme subunit. This regulation is triggered by binding of the aptamers target molecule, which causes significant structural changes in the aptamer and thus in the entire aptazyme. Therefore, aptazymes function similar to allosteric enzymes, whose catalytic activity is regulated by binding of ligands (effectors) to allosteric sites due to alteration of the three-dimensional structure of the active site of the enzyme. In case of aptazymes, the allosteric site is composed of an aptamer. Aptazymes can be designed for different applications and have already been used in analytical assays as well as for the regulation of gene expression.

Production of polycaprolactone nanoparticles with hydrodynamic diameters below 100 nm.

Cancer is a worldwide increasing burden and its therapy is often challenging and causes severe side effects in healthy tissue. If drugs are loaded into nanoparticles, side effects can be reduced, and efficiency can be increased via the enhanced permeability and retention effect. This effect is based on the fact that nanoparticles with sizes from 10 to 200 nm can accumulate in tumor tissue due to their leaky vasculature. In this work, we produced polycaprolactone (PCL) in the sizes 1.8, 5.4, and 13.6 kDa and were able to produce spherical shaped nanoparticles with mean diameters of 64 ± 19 nm out of the PCL5.4 and 45 ± 8 nm out of the PCL13.6 reproducibly. By encapsulation of paclitaxel the diameter of that nanoparticles did not increase, and we were able to encapsulate 73 ± 7 fmol paclitaxel per 1000 particles in the PCL5.4-nanoparticles and 35 ± 8 fmol PTX per 1000 PCL13.6-nanoparticles. Furthermore, we coupled the aptamer S15 to preformed PCL5.4-nanoparticles resulting in particles with a hydrodynamic diameter of 153 nm. This offers the opportunity to use these nanoparticles for targeted drug delivery.

Development of Aptamer-Based TID Assays Using Thermophoresis and Microarrays.

Aptamers are single-stranded oligonucleotides which can be used as alternative recognition elements for protein detection, because aptamers bind their targets with a high affinity similar to antibodies. Due to the targetinduced conformational changes of aptamers, these oligonucleotides can be applied in various biosensing platforms. In this work, aptamers directed against the vascular endothelial growth factor (VEGF) were used as a model system. VEGF plays a key role in physiological angiogenesis and vasculogenesis. Furthermore, VEGF is involved in the development and growth of cancer and other diseases like agerelated macular degeneration, rheumatoid arthritis, diabetes mellitus, and neurodegenerative disorders. Detecting the protein biomarker VEGF is therefore of great importance for medical research and diagnostics. In this research, VEGFbinding aptamers were investigated for the systematic development of a targetinduced dissociation (TID) assay utilizing thermophoresis and microarrays. The established aptamer-microarray allowed for the detection of 0.1 nM of VEGF. Furthermore, the systematic development of the TID method using the VEGF model protein could help to develop further TID assays for the detection of various protein biomarkers.

Systematic investigation of optimal aptamer immobilization for protein-microarray applications.

Aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to a wide range of target molecules with high affinity and specificity. As nucleic acids, aptamers can undergo denaturation, but the process is reversible. As a result of this stability and the possibility of automated selection of aptamers, these oligonucleotides are highly promising capture molecules in microarray formats. In this study, his-tagged proteins and an aptamer directed against the his-tag were chosen as a model system. Different factors affect the activity of aptamers immobilized on a solid support like a microarray surface. The orientation of the immobilized aptamer plays an important role in correct aptamer folding and, thus, in effective binding of the corresponding target. Other important parameters identified in this work are the microarrays' surface charge as well as the length of the spacer between aptamer and solid support. These parameters were investigated systematically, resulting in the development of an aptamer-based microarray for detection of his-tagged proteins. The general applicability of the developed immobilization strategy was demonstrated by utilization of three different aptamers.

Optimization of a microarray sandwich-ELISA against hINF-gamma on a modified nitrocellulose membrane.

The highly specific and highly sensitive ELISA (enzyme linked immunosorbent assay) technique is the most commonly used method for immunological diagnostics in general. In combination with protein microarrays and their ability to allow performing thousands of experiments in parallel, a promising tool for global analytical approaches with reduced consumption of time, analytes, and reagents is given. In this study a protein microarray-based sandwich-ELISA for human interferon-gamma (hINF-gamma) is established. In consideration of the immense importance of the surface chemistry, a new black nitrocellulose matrix that generates very high signal-to-noise ratios (SNR) and a very low autofluorescence was tested and optimized as microarray substrate. A validation of the applicability of the system was performed with a comparison to different commercially available systems. Experimental results show that the microarray-based ELISA is faster and easier to perform and shows a lower limit of detection (LOD) than a comparable system in a 96-well plate. The spotted slides with the capture antibody can be stored up to 1 month with no significant loss of signal intensity. A second model system with immobilized His-tagged restriction enzyme EcoRV and an anti-His antibody shows in coincidence the good applicability of the black nitrocellulose membrane and no cross-reactivity toward the ELISA.