Analytical applications of aptamers

Journal of Biomedical Optics, 2010

In this study, we exploit the sensitivity offered by surfaceenhanced Raman scattering ͑SERS͒ for the direct detection of thrombin using the thrombin-binding aptamer ͑TBA͒ as molecular receptor. The technique utilizes immobilized silver nanoparticles that are functionalized with thiolated thrombin-specific binding aptamer, a 15-mer ͑5Ј-GGTTGGTGTGGTTGG-3Ј͒ quadruplex forming oligonucleotide. In addition to the Raman vibrational bands corresponding to the aptamer and blocking agent, new peaks ͑mainly at 1140, 1540, and 1635 cm −1 ͒ that are characteristic of the protein are observed upon binding of thrombin. These spectral changes are not observed when the aptamer-nanoparticle assembly is exposed to a nonbinding protein such as bovine serum albumin ͑BSA͒. This methodology could be further used for the development of label-free biosensors for direct detection of proteins and other molecules of interest for which aptamers are available.

Electroanalysis, 2012

We report an aptamer biosensing array for thrombin detection by measuring the electrochemical impedance upon aptamer-protein formation at the surface of CD-trodes (GCDTs) in the presence of the redox couple [Fe(CN) 6 ] 3À/4À . GCDTs are fabricated from recordable compact discs that contain a fine gold layer. The biosensor is constructed by self-assembling of a thiol-modified thrombin binding aptamer (TBA) onto a GCDT surface. The sensor reveals good ligand specificity, recognition in a wide range of thrombin concentrations from 20 nM to 1 mM with a limit of detection of 5 nM.

Nanotechnology, 2014

Atomic force microscopy-based adhesion force measurements have emerged as a powerful tool for the biophysical analyses of biological systems. Such measurements can now be extended to detection and mapping of biomolecules on surfaces via integrated imaging and force spectroscopy techniques. Critical to these experiments is the choice of the biomolecular recognition probe. In this study, we demonstrate how oligonucleotide aptamers can be used as versatile probes to simultaneously image and spatially locate targets on surfaces. We focus on two structurally distinct proteins relevant to the clotting cascade-human α-thrombin and vascular endothelial growth factor. Via AFM-recognition mapping using specific DNA aptamers on a commercially available instrument, we show a clear consistency between height and force measurements obtained simultaneously. Importantly, we are able to observe changes in binding due to changes in the external microenvironment, which demonstrate the ability to study fluctuating biological systems in real time. The aptamer specificity and the ability to distinguish their targets are shown through positive and negative controls. It is therefore possible to generate high resolution maps to spatially and temporally identify proteins at the molecular level on complex surfaces.

PLOS ONE, 2015

Malignant glioma is characterised by a rapid growth rate and high capacity for invasive infiltration to surrounding brain tissue; hence, diagnosis and treatment is difficult and patient survival is poor. Aptamers contribute a promising and unique technology for the in vitro imaging of live cells and tissues, with a potentially bright future in clinical diagnostics and therapeutics for malignant glioma. The binding selectivity, uptake capacity and binding target of two DNA aptamers, SA43 and SA44, were investigated in glioma cells and patient tissues. The binding assay showed that SA43 and SA44 bound with strong affinity (K d , 21.56 ± 4.60 nM and K d , 21.11 ± 3.30 nM respectively) to the target U87MG cells. Quantitative analysis by flow cytometry showed that the aptamers were able to actively internalise in U87MG and 1321N1 glioma cells compared to the non-cancerous and non-glioma cell types. Confocal microscopy confirmed staining in the cytoplasm, and co-localisation studies with endoplasmic reticulum, Golgi apparatus and lysosomal markers suggested internalisation and compartmentalisation within the endomembrane system. Both aptamers selectively bound to Ku 70 and Ku 80 DNA repair proteins as determined by aptoprecipitation (AP) followed by mass spectrometry analysis and confirmation by Western blot. In addition, aptohistochemical (AHC) staining on paraffin embedded, formalin fixed patient tissues revealed that the binding selectivity was significantly higher for SA43 aptamer in glioma tissues (grade I, II, III and IV) compared to the non-cancerous tissues, whereas SA44 did not show selectivity towards glioma tissues. The results indicate that SA43 aptamer can differentiate between glioma and non-cancerous cells and tissues and therefore, shows promise for histological diagnosis of glioma.

Sensors, 2009

Nanomaterials have become one of the most interesting sensing materials because of their unique size-and shape-dependent optical properties, high surface energy and surface-to-volume ratio, and tunable surface properties. Aptamers are oligonucleotides that can bind their target ligands with high affinity. The use of nanomaterials that are bioconjugated with aptamers for selective and sensitive detection of analytes such as small molecules, metal ions, proteins, and cells has been demonstrated. This review focuses on recent progress in the development of biosensors by integrating functional aptamers with different types of nanomaterials, including quantum dots, magnetic nanoparticles (NPs), metallic NPs, and carbon nanotubes. Colorimetry, fluorescence, electrochemistry, surface plasmon resonance, surface-enhanced Raman scattering, and magnetic resonance imaging are common detection modes for a broad range of analytes with high sensitivity and selectivity when using aptamer bioconjugated nanomaterials (Apt-NMs). We highlight the important roles that the size and concentration of nanomaterials, the secondary structure and density of aptamers, and the multivalent interactions play in determining the specificity and sensitivity of the nanosensors towards analytes. Advantages and disadvantages of the Apt-NMs for bioapplications are focused.

Analytical and Bioanalytical Chemistry, 2011

Native C-reactive protein (CRP) is composed of five identical subunits arranged in a pentameric structure (pCRP). Binding of pCRP to damaged cell membranes produces a second isoform, modified CRP, which has similar antigenicity to isolated monomeric subunits of CRP (mCRP). Emerging evidence indicates that modified CRP plays a role in inflammation and atherosclerosis, however, there are very few techniques that can distinguish the different isoforms of CRP. Here we show that an RNA aptamer binds specifically to mCRP and not to pCRP. Using this aptamer, we describe a simple, fast, and sensitive assay to detect nanomolar concentrations of mCRP using fluorescence anisotropy. In addition, we show that this aptamer can be used to detect mCRP in polyacrylamide gels and bound to a surface using total internal reflection fluorescence microscopy. The biological activity of the mCRP we prepared by heating pCRP with 0.1% sodium dodecyl sulfate was confirmed by observing binding to the complement protein, C1q. This probe provides an important tool for CRP research and has the potential to improve clinical diagnostics that predict risk for cardiovascular disease.

Sensors, 2008

Quantification of biological or biochemical processes are of utmost importance for medical, biological and biotechnological applications. However, converting the biological information to an easily processed electronic signal is challenging due to the complexity of connecting an electronic device directly to a biological environment. Electrochemical biosensors provide an attractive means to analyze the content of a biological sample due to the direct conversion of a biological event to an electronic signal. Over the past decades several sensing concepts and related devices have been developed. In this review, the most common traditional techniques, such as cyclic voltammetry, chronoamperometry, chronopotentiometry, impedance spectroscopy, and various field-effect transistor based methods are presented along with selected promising novel approaches, such as nanowire or magnetic nanoparticle-based biosensing. Additional measurement techniques, which have been shown useful in combination with electrochemical detection, are also summarized, such as the electrochemical versions of surface plasmon resonance, optical waveguide lightmode spectroscopy, ellipsometry, quartz crystal microbalance, and scanning probe microscopy. The signal transduction and the general performance of electrochemical sensors are often determined by the surface architectures that connect the sensing element to the biological sample at the nanometer scale. The most common surface modification techniques, the various electrochemical transduction mechanisms, and the choice of the recognition receptor molecules all influence the ultimate sensitivity of the sensor. New nanotechnology-based approaches, such as the use of engineered ion-channels in lipid bilayers, the encapsulation of enzymes into vesicles, polymersomes, or polyelectrolyte capsules provide additional possibilities for signal amplification. In particular, this review highlights the importance of the precise control over the delicate interplay between surface nano-architectures, surface functionalization and the chosen sensor transducer principle, as well as the usefulness of complementary characterization tools to interpret and to optimize the sensor response.

Sensors (Basel, Switzerland), 2015

Nanoscale biosensors provide the possibility to miniaturize optic, acoustic and electric sensors to the dimensions of biomolecules. This enables approaching single-molecule detection and new sensing modalities that probe molecular conformation. Nanoscale sensors are predominantly surface-based and label-free to exploit inherent advantages of physical phenomena allowing high sensitivity without distortive labeling. There are three main criteria to be optimized in the design of surface-based and label-free biosensors: (i) the biomolecules of interest must bind with high affinity and selectively to the sensitive area; (ii) the biomolecules must be efficiently transported from the bulk solution to the sensor; and (iii) the transducer concept must be sufficiently sensitive to detect low coverage of captured biomolecules within reasonable time scales. The majority of literature on nanoscale biosensors deals with the third criterion while implicitly assuming that solutions developed for ma...

Electroanalysis, 2009

Aptamer hybridizes with its complementary strand. However, the complementary strand has difficulties to hybridize with the aptamer bound to a target because the aptamer forms higher-order structures. Exploiting this property, we developed simple bound/free separation systems for thrombin and IgE detection. The complementary strand was immobilized onto beads and the aptamer was labeled with pyrroquinoline quinone glucose dehydrogenase (PQQGDH). In the absence of a target, the aptamer is trapped by beads, whereas in the presence of a target, the aptamer bound to the target is not trapped. Thus the aptamer-target complexes can be recovered easily and detected by PQQGDH activity. This system allow the detection of 270 pM thrombin and 1 nM IgE.

Chemical Communications, 2011

Device fabrication protocol followed in this work was adapted from procedure described previously. 1 A uniformly dispersed and separated SWNTs suspension was prepared by ultrasonication (power level 9) and centrifugation (10,000 rpm) a 25 μg SWNTs (high carboxylated functionality, sold under the trade name of P3-SWNT, were purchased from Carbon Solutions, Inc. Riverside, CA, USA) in 1 mL DMF for three different times (90, 60, 30 min for each cycle). A 0.1 µL drop of the dispersed SWNTs was dispensed between the gap (3 μm) of a pair of microfabricated gold electrodes and aligned by AC dielectrophoresis (DEP) by applying a 4 MHz (amplitude 0.366V p-p) AC field across the electrodes for a few seconds using a function generator (Wavetek, Alpharetta, GA, USA) and then annealed at 300 o C for an hour under a continuous flow of 5% H 2 plus 95% nitrogen gas to evaporate residual DMF and reduce the contact resistance between the carbon nanotubes and the gold electrodes. The SWNTs interconnects/channel of the chemiresistor was then incubated with 6 mM 1-pyrenebutanoic acid succinimidyl ester (PBASE) in DMF for 1 h followed by thorough washing with DMF to remove residual esters. The capture oligonucleotides were covalently attached to the PBASE-modified SWNTs by incubating overnight at 4 o C 200 nM oligo (5'-/5AmMC6/TGT GAC ACC TTC CC-3') or aptamer (5'-ACC TGG GGG AGT ATT GCG GAG GAA GGT GTC ACA-3' for the traditional format) in 10 mM pH 7.2 phosphate buffer (PB) through the amide bond between the amine at its 5' end and Nhydrosuccinimide ester of PBASE washed three times with PB to remove excess oligos followed by treatment with 0.1 mM ethanolamine for 30 min at room temperature to block excessive reactive groups and finally by incubation with 0.1% Tween 20 to prevent nonspecific binding. The capture oligos were subsequently hybridized to aptamers by incubating with a 200 nM aptamer solution in PB heated at 65 0 C for 5 min, then for 2 h at room temperature and washed with PB 3 times to remove excess aptamers.

ACS Nano

Lateral flow assays (LFAs) are paper-based point-of-care (POC) diagnostic tools that are widely used because of their low cost, ease of use, and rapid format. Unfortunately, traditional commercial LFAs have significantly poorer sensitivities (μM) and specificities than standard laboratory tests (enzyme-linked immunosorbent assay, ELISA: pM−fM; polymerase chain reaction, PCR: aM), thus limiting their impact in disease control. In this Perspective, we review the evolving efforts to increase the sensitivity and specificity of LFAs. Recent work to improve the sensitivity through assay improvement includes optimization of the assay kinetics and signal amplification by either reader systems or additional reagents. Together, these efforts have produced LFAs with ELISA-level sensitivities (pM−fM). In addition, sample preamplification can be applied to both nucleic acids (direct amplification) and other analytes (indirect amplification) prior to LFA testing, which can lead to PCR-level (aM) sensitivity. However, these amplification strategies also increase the detection time and assay complexity, which inhibits the large-scale POC use of LFAs. Perspectives to achieve future rapid (<30 min), ultrasensitive (PCR-level), and "sample-to-answer" POC diagnostics are also provided. In the case of LFA specificity, recent research efforts have focused on high-affinity molecules and assay optimization to reduce nonspecific binding. Furthermore, novel highly specific molecules, such as CRISPR/Cas systems, can be integrated into diagnosis with LFAs to produce not only ultrasensitive but also highly specific POC diagnostics. In summary, with continuing improvements, LFAs may soon offer performance at the POC that is competitive with laboratory techniques while retaining a rapid format. A lthough infectious diseases have always posed global threats, there is no clearer current example of the need for inexpensive and high-performing (i.e., low rates of false negatives (FNs) and positives (FPs)) point-of-care (POC) diagnostics than with the global pandemic of SARS-CoV-2 (i.e., COVID-19). As a single serological antibody or antigen test can only indicate past or recent exposure to SARS-CoV-2, multiple and broad testing throughout the population will be needed to identify "hot spots" and to control the disease effectively. 1−5 In an ideal situation, at-risk individuals would be tested regularly (i.e., weekly or daily) to enable timely isolation and to minimize virus transmission among the community. 1−5 Unfortunately, this need cannot be met using the current primary diagnostic tools, such as reverse transcription polymerase chain reaction (RT-PCR). Although RT-PCR has excellent sensitivity and specificity, it cannot be used as a POC test because this method requires trained staff in laboratories equipped with specialized thermal cycling equipment and strict environmental conditions to prevent contamination. 6−8 In addition, the long turnaround time (hours to days) and high cost of RT-PCR (100−200 USD per COVID-19 swab test) compared to other rapid (<15 min) diagnostic tools, such as lateral flow assays (LFAs; <$50 per COVID-19 swab test), 9,10 restrict its deployment in POC settings. These costs can be expected to drop further because other commercialized LFAs, such as human chorionic gonadotropin (pregnancy) LFAs, are <$1 per test. Because LFAs are arguably the cheapest, fastest, and easiest to use paper-based POC tests, 11−15 they exhibit promise as a tool for achieving global pandemic control by enabling the rapid screening of infections. In addition to the detection of SARS-CoV-2, LFAs have also been widely applied in biomedicine, food contaminant and toxic chemical detection, and environmental monitoring. 12 In biomedical diagnosis, an important advantage of this technology is that it enables the decentralization of laboratory testing to POC sites. Some important examples of LFAs are those used for the rapid diagnosis of influenza, 16,17 Streptococcus, 18,19 and many other viral and bacterial infections. 20−22 The Centers for Disease

Food Additives & Contaminants: Part A, 2011

This review examines the developments in optical biosensor technology, which uses the phenomenon of surface plasmon resonance, for the detection of paralytic shellfish poisoning (PSP) toxins. Optical biosensor technology measures the competitive biomolecular interaction of a specific biological recognition element or binder with a target toxin immobilised onto a sensor chip surface against toxin in a sample. Different binders such as receptors and antibodies previously employed in functional and immunological assays have been assessed. Highlighted are the difficulties in detecting this range of low molecular weight toxins, with analogues differing at four chemical substitution sites, using a single binder. The complications that arise with the toxicity factors of each toxin relative to the parent compound, saxitoxin, for the measurement of total toxicity relative to the mouse bioassay are also considered. For antibodies, the cross-reactivity profile does not always correlate to toxic potency, but rather to the toxin structure to which it was produced. Restrictions and availability of the toxins makes alternative chemical strategies for the synthesis of protein conjugate derivatives for antibody production a difficult task. However, when two antibodies with different cross-reactivity profiles are employed, with a toxin chip surface generic to both antibodies, it was demonstrated that the cross-reactivity profile of each could be combined into a single-assay format. Difficulties with receptors for optical biosensor analysis of low molecular weight compounds are discussed, as are the potential of alternative non-antibody-based binders for future assay development in this area.

PLOS ONE, 2016

An increase in the expression of estrogen receptors (ER) and the expanded population of ER-positive cells are two common phenotypes of breast cancer. Detection of the aberrantly expressed ERα in breast cancer is carried out using ERα-antibodies and radiolabelled ligands to make decisions about cancer treatment and targeted therapy. Capitalizing on the beneficial advantages of aptamer over the conventional antibody or radiolabelled ligand, we have identified a DNA aptamer that selectively binds and facilitates the detection of ERα in human breast cancer tissue sections. The aptamer is identified using the high throughput sequencing assisted SELEX screening. Biophysical characterization confirms the binding and formation of a thermodynamically stable complex between the identified DNA aptamer (ERaptD4) and ERα (Ka = 1.55±0.298×10 8 M-1 ; ΔH = 4.32×10 4 ±801.1 cal/mol; ΔS =-108 cal/mol/deg). Interestingly, the specificity measurements suggest that the ERaptD4 internalizes into ERα-positive breast cancer cells in a target-selective manner and localizes specifically in the nuclear region. To harness these characteristics of ERaptD4 for detection of ERα expression in breast cancer samples, we performed the aptamer-assisted histochemical analysis of ERα in tissue samples from breast cancer patients. The results were validated by performing the immunohistochemistry on same samples with an ERα-antibody. We found that the two methods agree strongly in assay output (kappa value = 0.930, pvalue <0.05 for strong ERα positive and the ERα negative samples; kappa value = 0.823, pvalue <0.05 for the weak/moderate ER+ve samples, n = 20). Further, the aptamer stain the ERα-positive cells in breast tissues without cross-reacting to ERα-deficient fibroblasts, adipocytes, or the inflammatory cells. Our results demonstrate a significant consistency in the aptamer-assisted detection of ERα in strong ERα positive, moderate ERα positive and ERα negative breast cancer tissues. We anticipate that the ERaptD4 aptamer targeting ERα may potentially be used for an efficient grading of ERα expression in cancer tissues.

Analytical and Bioanalytical Chemistry, 2011

A new solid phase extraction method based on aptamers, an oligosorbent, was developed and applied to the determination of ochratoxin A (OTA) from red wine. Two solid supports were chosen to immobilize OTA aptamer by covalent binding (cyanogen bromide-activated sepharose) or noncovalent binding (streptavidin-activated agarose). The resulting oligosorbents were evaluated in terms of retention, selectivity, and capacity. To assess the selectivity of the resulting oligosorbents, control supports made only of a solid support without immobilized aptamers were simultaneously studied. After optimization of the selective extraction procedure, extraction recoveries close to 100% were obtained on both materials. No retention was observed on the control supports. A similar capacity was also found for both oligosorbents. However, the immobilization by covalent bonding appeared more robust for the determination of OTA in the wine. A conventional sorbent and an immunoaffinity column were also applied to the determination of OTA in red wine to compare the potential of the various approaches for the treatment of such complex samples.

Expert Opinion on Drug Discovery, 2007

Nanoscale Advances

We describe how magnetic nanoparticles can be used to study intracellular nanoparticle trafficking, and how magnetic extraction may be integrated with downstream analyses to investigate nanoscale decision-making events.

Biomedicines

Selected reaction monitoring (SRM) is a mass spectrometric technique characterized by the exceptionally high selectivity and sensitivity of protein detection. However, even with this technique, the quantitative detection of low- and ultralow-abundance proteins in blood plasma, which is of great importance for the search and verification of novel protein disease markers, is a challenging task due to the immense dynamic range of protein abundance levels. One approach used to overcome this problem is the immunoaffinity enrichment of target proteins for SRM analysis, employing monoclonal antibodies. Aptamers appear as a promising alternative to antibodies for affinity enrichment. Here, using recombinant protein SMAD4 as a model target added at known concentrations to human blood plasma and SRM as a detection method, we investigated a relationship between the initial amount of the target protein and its amount in the fraction enriched with SMAD4 by an anti-SMAD4 DNA-aptamer immobilized o...

Biomedicines, 2015

Aptamers are nucleic acid/peptide molecules that can be generated by a sophisticated, well-established technique known as Systematic Evolution of Ligands by EXponential enrichment (SELEX). Aptamers can interact with their targets through structural recognition, as in antibodies, though with higher specificity. With this added advantage, they can be made useful for clinical applications such as targeted therapy and diagnosis. In this review, we have discussed the steps involved in SELEX process and modifications executed to attain high affinity nucleic acid aptamers. Moreover, our review also highlights the therapeutic applications of aptamer functionalized nanoparticles and nucleic acids as chemo-therapeutic agents. In addition, we have described the development of &quot;aptasensor&quot; in clinical diagnostic application for detecting cancer cells and the use of aptamers in different routine imaging techniques, such as Positron Emission Tomography/Computed Tomography, Ultrasound, a...

Sensors, 2012

Recently, aptamers have attracted the attention of many scientists, because they not only have all of the advantages of antibodies, but also have unique merits, such as thermal stability, low cost, and unlimited applications. In this review, we present the reasons why aptamers are known as alternatives to antibodies. Furthermore, several types of in vitro selection processes, including nitrocellulose membrane filtration, affinity chromatography, magnetic bead, and capillary electrophoresis-based selection methods, are explained in detail. We also introduce various applications of aptamers for the diagnosis of diseases and detection of small molecules. Numerous analytical techniques, such as electrochemical, colorimetric, optical, and mass-sensitive methods, can be utilized to detect targets, due to convenient modifications and the stability of aptamers. Finally, several medical and analytical applications of aptamers are presented. In summary, aptamers are promising materials for diverse areas, not just as alternatives to antibodies, but as the core components of medical and analytical equipment.

Nutrients

Celiac disease, wheat sensitivity, and allergy represent three different reactions, which may occur in genetically predisposed individuals on the ingestion of wheat and derived products with various manifestations. Improvements in the disease diagnostics and understanding of disease etiology unveiled that these disorders are widespread around the globe affecting about 7% of the population. The only known treatment so far is a life-long gluten-free diet, which is almost impossible to follow because of the contamination of allegedly “gluten-free” products. Accidental contamination of inherently gluten-free products could take place at any level from field to shelf because of the ubiquity of these proteins/grains. Gluten contamination of allegedly “gluten-free” products is a constant threat to celiac patients and a major health concern. Several detection procedures have been proposed to determine the level of contamination in products for celiac patients. The present article aims to re...

Technologies

Hydrogels, swellable hydrophilic polymer networks fabricated through chemical cross-linking or physical entanglement are increasingly utilized in various biomedical applications over the past few decades. Hydrogel-based microparticles, dressings and microneedle patches have been explored to achieve safe, sustained and on-demand therapeutic purposes toward numerous skin pathologies, through incorporation of stimuli-responsive moieties and therapeutic agents. More recently, these platforms are expanded to fulfill the diagnostic and monitoring role. Herein, the development of hydrogel technology to achieve diagnosis and monitoring of pathological skin conditions are highlighted, with proteins, nucleic acids, metabolites, and reactive species employed as target biomarkers, among others. The scope of this review includes the characteristics of hydrogel materials, its fabrication procedures, examples of diagnostic studies, as well as discussion pertaining clinical translation of hydrogel ...

Sensors (Basel, Switzerland), 2016

Various methodologies have been reported in the literature for the qualitative and quantitative monitoring of mycotoxins in food and feed samples. Based on their enhanced specificity, selectivity and versatility, bio-affinity assays have inspired many researchers to develop sensors by exploring bio-recognition phenomena. However, a significant problem in the fabrication of these devices is that most of the biomolecules do not generate an easily measurable signal upon binding to the target analytes, and signal-generating labels are required to perform the measurements. In this context, aptamers have been emerged as a potential and attractive bio-recognition element to design label-free aptasensors for various target analytes. Contrary to other bioreceptor-based approaches, the aptamer-based assays rely on antigen binding-induced conformational changes or oligomerization states rather than binding-assisted changes in adsorbed mass or charge. This review will focus on current designs i...

Next Generation Point-of-care Biomedical Sensors Technologies for Cancer Diagnosis

Sensors

The urgent need to develop a detection system for Staphylococcus aureus, one of the most common causes of infection, is prompting research towards novel approaches and devices, with a particular focus on point-of-care analysis. Biosensors are promising systems to achieve this aim. We coupled the selectivity and affinity of aptamers, short nucleic acids sequences able to recognize specific epitopes on bacterial surface, immobilized at high density on a nanostructured zirconium dioxide surface, with the rational design of specifically interacting fluorescent peptides to assemble an easy-to-use detection device. We show that the displacement of fluorescent peptides upon the competitive binding of S. aureus to immobilized aptamers can be detected and quantified through fluorescence loss. This approach could be also applied to the detection of other bacterial species once aptamers interacting with specific antigens will be identified, allowing the development of a platform for easy detec...

Scientific Reports

Different lines of evidence indicate that monitoring the blood levels of therapeutic antibodies, characterized by high inter-individual variability, can help to optimize clinical decision making, improving patient outcomes and reducing costs with these expensive treatments. A surface plasmon resonance (SPR)-based immunoassay has recently been shown to allow highly reliable and robust monitoring of serum concentrations of infliximab, with significant advantages over classical ELISA. The next level of advancement would be the availability of compact and transportable SPR devices suitable for easy, fast and cheap point-of-care analysis. Here we report the data obtained with recently developed, cost-effective, optical-fibre-based SPR sensors (SPR-POF), which allow the construction of a compact miniaturized system for remote sensing. We carried out an extensive characterization of infliximab binding to an anti-infliximab antibody immobilized on the SPR-POF sensor surface. The present pro...

Toxins, 2016

Since ochratoxin A (OTA) was discovered, it has been ubiquitous as a natural contaminant of moldy food and feed. The multiple toxic effects of OTA are a real threat for human beings and animal health. For example, OTA can cause porcine nephropathy but can also damage poultries. Humans exposed to OTA can develop (notably by inhalation in the development of acute renal failure within 24 h) a range of chronic disorders such as upper urothelial carcinoma. OTA plays the main role in the pathogenesis of some renal diseases including Balkan endemic nephropathy, kidney tumors occurring in certain endemic regions of the Balkan Peninsula, and chronic interstitial nephropathy occurring in Northern African countries and likely in other parts of the world. OTA leads to DNA adduct formation, which is known for its genotoxicity and carcinogenicity. The present article discusses how renal carcinogenicity and nephrotoxicity cause both oxidative stress and direct genotoxicity. Careful analyses of the data show that OTA carcinogenic effects are due to combined direct and indirect mechanisms (e.g., genotoxicity, oxidative stress, epigenetic factors). Altogether this provides strong evidence that OTA carcinogenicity can also occur in humans.

Sensors

This paper focuses on the current state of art as well as on future trends in electrochemical aptasensors application in medical diagnostics. The origin of aptamers is presented along with the description of the process known as SELEX. This is followed by the description of the broad spectrum of aptamer-based sensors for the electrochemical detection of various diagnostically relevant analytes, including metal cations, abused drugs, neurotransmitters, cancer, cardiac and coagulation biomarkers, circulating tumor cells, and viruses. We described also possible future perspectives of aptasensors development. This concerns (i) the approaches to lowering the detection limit and improvement of the electrochemical aptasensors selectivity by application of the hybrid aptamer–antibody receptor layers and/or nanomaterials; and (ii) electrochemical aptasensors integration with more advanced microfluidic devices as user-friendly medical instruments for medical diagnostic of the future.

Chemical Communications, 2011

Material and Reagents. (1-Mercaptoundec-11-yl)tetra(ethyleneglycol) (95%) was purchased from Sigma-Aldrich (St. Louis, MO). Molecular biology grade water was purchase from Fisher Scientific. All other chemicals were of analytical grade and used without any further purification. Preparation of SERS-Active Ag Nanorod Arrays. Ag nanorod array substrates were prepared by an oblique-angle vapor deposition (OAD) technique using a custom-designed electron-beam/sputtering evaporation system. 1, 2 Standard 2.54  7.62 cm glass microscope slides (Gold Seal ® , Becton Dickinson Company, Franklin Lakes, NJ) were cleaned using Piranha solution (4:1 conc. H 2 SO 4 : 30% H 2 O 2) for 15 minutes, rinsed several times with copious amounts of deionized water and dried under a gentle stream of nitrogen gas. The glass slides were placed into a vapor deposition chamber with background pressure maintained at less than 5  10-6 Torr. A 20 nm layer of Ti (99.99%, Kurt J. Lesker Company, Clairton, PA) was deposited onto the glass slide substrate at a rate no greater than 2.0 Å/s. The Ti underlayer serves as an adhesion layer on the glass slide to stabilize the subsequent Ag layers. Next, a 500 nm layer of Ag (>99.99%, Kurt J. Lesker Company) was deposited onto the substrate at a rate of 3.0-4.0 Å/s. Finally, the substrate was rotated to 86° relative to the incident vapor source. Ag nanorods were then deposited at a constant rate of 2.0-3.0 Å/s until a quartz crystal microbalance (QCM) registered a final nominal thickness of 2000 nm. The QCM was placed inside the deposition chamber and positioned at normal incidence to the vapor source. We have established a calibration relationship between nanorod length and the mass of Ag deposited on the quartz crystal where 2000 nm nominal thickness results in tilted nanorods of ~900 nm length. After deposition, the SERS-active substrates were allowed to cool down to room temperature in vacuum inside the deposition chamber for 2 hours. The SERS-active substrates were then removed from the deposition chamber and stored in a nitrogen purge type glove box

LI:BIOSENS NANOMATERIALS O-BK, 2011

Pharmaceuticals

The current Covid-19 pandemic has pointed out some major deficiencies of the even most advanced societies to fight against viral RNA infections. Once more, it has been demonstrated that there is a lack of efficient drugs to control RNA viruses. Aptamers are efficient ligands of a great variety of molecules including proteins and nucleic acids. Their specificity and mechanism of action make them very promising molecules for interfering with the function encoded in viral RNA genomes. RNA viruses store essential information in conserved structural genomic RNA elements that promote important steps for the consecution of the infective cycle. This work describes two well documented examples of RNA aptamers with antiviral activity against highly conserved structural domains of the HIV-1 and HCV RNA genome, respectively, performed in our laboratory. They are two good examples that illustrate the potential of the aptamers to fill the therapeutic gaps in the fight against RNA viruses.

Polymers, 2018

Nucleic acid therapeutics have the potential to be the most effective disease treatment strategy due to their intrinsic precision and selectivity for coding highly specific biological processes. However, freely administered nucleic acids of any type are quickly destroyed or rendered inert by a host of defense mechanisms in the body. In this work, we address the challenge of using nucleic acids as drugs by preparing stimuli responsive poly(methacrylic acid)/poly(N-vinylpyrrolidone) (PMAA/PVPON)n multilayer hydrogel capsules loaded with ~7 kDa G-quadruplex DNA. The capsules are shown to release their DNA cargo on demand in response to both enzymatic and ultrasound (US)-triggered degradation. The unique structure adopted by the G-quadruplex is essential to its biological function and we show that the controlled release from the microcapsules preserves the basket conformation of the oligonucleotide used in our studies. We also show that the (PMAA/PVPON) multilayer hydrogel capsules can ...

Journal of Sol-Gel Science and Technology, 2018

In recent years RNA aptamers have emerged as potential recognition elements for solid phase assays, including assays that utilize sol-gel based biohybrid materials. However, there is still very little knowledge regarding the behavior of RNA aptamers when entrapped in sol-gel-derived materials. In this work, we evaluated the performance of an adenosine triphosphate (ATP)-binding structure-switching RNA aptamer in a series of sol-gel derived materials and compared the results to those previously reported for an ATP-binding DNA aptamer. It was observed that the nature of the entrapping material is the key parameter affecting the functionality of the entrapped ATP-binding RNA aptamer, which mainly impacts its ability to remain fully hybridized to signaling DNA strands upon entrapment. We observed that those materials with a high organic content provided the best performance for entrapped RNA aptamers at early times after entrapment. However, upon aging, materials derived from sodium silicate provided the best performance. Overall, the results suggest that polar materials that do not produce alcohol are optimal for entrapment of both DNA and RNA aptamers that bind ATP.

International Journal of Molecular Sciences, 2008

Aptamers, single stranded DNA or RNA molecules, generated by a method called SELEX (systematic evolution of ligands by exponential enrichment) have been widely used in various biomedical applications. The newly developed Cell-SELEX (cell based-SELEX) targeting whole living cells has raised great expectations for cancer biology, -therapy and regenerative medicine. Combining nanobiotechnology with aptamers, this technology opens the way to more sophisticated applications in molecular diagnosis. This paper gives a review of recent developments in SELEX technologies and new applications of aptamers.

Cancers

Cancer is the second cause of mortality worldwide. Early diagnosis of this multifactorial disease is challenging, especially in populations with limited access to healthcare services. A vast repertoire of cancer biomarkers has been studied to facilitate early diagnosis; particularly, the use of antibodies against these biomarkers has been of interest to detect them through biorecognition. However, there are certain limitations to this approach. Emerging biorecognition engineering technologies are alternative methods to generate molecules and molecule-based scaffolds with similar properties to those presented by antibodies. Molecularly imprinted polymers, recombinant antibodies, and antibody mimetic molecules are three novel technologies commonly used in scientific studies. This review aimed to present the fundamentals of these technologies and address questions about how they are implemented for cancer detection in recent scientific studies. A systematic analysis of the scientific p...

International Journal of Molecular Sciences

The over-growth and coagulation of nanoparticles is prevented using capping agents by the production of stearic effect that plays a pivotal role in stabilizing the interface. This strategy of coating the nanoparticles’ surface with capping agents is an emerging trend in assembling multipurpose nanoparticles that is beneficial for improving their physicochemical and biological behavior. The enhancement of reactivity and negligible toxicity is the outcome. In this review article, an attempt has been made to introduce the significance of different capping agents in the preparation of nanoparticles. Most importantly, we have highlighted the recent progress, existing roadblocks, and upcoming opportunities of using surface modified nanoparticles in nanomedicine from the drug and gene delivery, bioimaging, and biosensing perspectives.

Biosensors, 2018

Food and environmental monitoring is one of the most important aspects of dealing with recent threats to human well-being and ecosystems. In this framework, electrochemical aptamer-based sensors are resilient due to their ability to resolve food and environmental contamination. An aptamer-based sensor is a compact analytical device combining an aptamer as the bio-sensing element integrated on the transducer surface. Aptamers display many advantages as biorecognition elements in sensor development when compared to affinity-based (antibodies) sensors. Aptasensors are small, chemically unchanging, and inexpensive. Moreover, they offer extraordinary elasticity and expediency in the design of their assemblies, which has led to innovative sensors that show tremendous sensitivity and selectivity. This review will emphasize recent food and environmental safeguarding using aptasensors; there are good prospects for their performance as a supplement to classical techniques.

Toxins, 2018

This work focuses on the development of the novel label-free optical apta-sensors for detection of mycotoxins. A highly sensitive analytical method of total internal reflection ellipsometry (TIRE) combined with Localized Surface Plasmon Resonance (LSPR) phenomenon in nano-structured gold films was exploited here for the first time for detection of aflatoxin B1 and M1 in direct assay with specific aptamers immobilized on the surface of gold. The achieved detection of low molecular weight molecules, such as aflatoxin B1 and M1, in a wide range of concentrations from 100 ng/mL down to 0.01 ng/mL is remarkable for the LSPR method. The study of binding kinetics of aflatoxin molecules to their respective aptamers using dynamic TIRE measurements yielded the values of affinity constants in the range of 10−8–10−7 mol, which is characteristic for highly specific aptamer/target interactions similar to that for monoclonal antibodies. The effect of aptamers’ DNA chain length on their binding cha...

European Journal of Clinical Microbiology &amp; Infectious Diseases, 2016

Haemophilus influenzae type b (Hib) causes acute bacterial meningitis (ABM) in children, with a mortality rate of about 3-6 % of the affected patients. ABM can lead to death during a period of hours to several days and, hence, rapid and early detection of the infection is crucial. Aptamers, the short single-stranded DNA or RNA with high affinity to target molecules, are selected by a high-flux screening technique known as in vitro screening and systematic evolution of ligands by exponential enrichment technology (SELEX). In this study, whole-cell SELEX was applied for the selection of targetspecific aptamers with high affinity to Hib. ssDNA aptamers prepared by lambda exonuclease were incubated with the target cells (Hib). The aptameric binding rate to Hib was characterized for binding affinity after seven SELEX rounds by flow cytometry. The aptamers with higher binding affinity were cloned. Four of 68 aptamer clones were selected for sequencing. The dissociation constant (K d) of the high-affinity aptamer clones 45 and 63 were 47.10 and 28.46 pM, respectively. These aptamers did not bind to other bacterial species, including the seven meningitis-causing bacteria. They showed distinct affinity to various H. influenzae strains only. These aptamers showed the highest affinity to Hib and the lowest affinity to H. influenzae type c and to other meningitis-causing bacteria. Clone 63 could detect Hib in patients' cerebrospinal fluid (CSF) samples at 60 colony-forming units (CFU)/mL. The results indicate applicability of the aptamers for rapid and early detection of infections brought about by Hib.

Scientific Reports, 2019

The development of optical biosensors for the rapid and costless determination of clinical biomarkers is of paramount importance in medicine. Here we report a fast and low-cost biosensor based on a plasmonic D-shaped plastic optical fibre (POF) sensor derivatized with an aptamer specific for the recognition of thrombin, the target marker of blood homeostasis and coagulation cascade. In particular, we designed a functional interface based on a Self Assembled Monolayer (SAM) composed of short Poly Ethylene Glycol (PEG) chains and biotin-modified PEG thiol in ratio 8:2 mol:mol, these latter serving as baits for the binding of the aptamer through streptavidin-chemistry. The SAM was studied by X-ray Photoelectron Spectroscopy (XPS) analysis, static contact angle (CA), Surface Plasmon Resonance (SPR) in POFs, and fluorescence microscopy on gold surface. The optimized SAM composition enabled the immobilization of about 112 ng/cm2 of aptamer. The thrombin detection exploiting POF-Aptasensor...

Food Analytical Methods, 2019

Mepanipyrim is a new-generation anilinopyrimidine pesticide that is employed nowadays to fight fungal diseases in a variety of crops. Rapid and user-friendly analytical methods that can complement the instrumental techniques are demanded. To analyse residues of this toxic compound in foodstuffs, monoclonal antibodies were generated for the first time, and an enzymelinked immunosorbent assay was developed. In the present study, a series of haptens mimicking the mepanipyrim molecule, and with a linker at alternative sites of the molecular framework, were designed and prepared by total synthesis in order to study the influence of the linker tethering site over the binding properties of the generated antibodies. This strategy afforded high-affinity and specific monoclonal antibodies to mepanipyrim. Interestingly, the highest sensitivity was reached with antibodies that had been raised using haptens with the spacer arm at the most characteristic moiety of the analyte, the 1-propynyl group. Moreover, the influence of the triple bond was investigated both in immunizing bioconjugates and in assay antigens. Finally, a competitive enzyme-linked immunosorbent assay was optimized and characterized, showing an IC 50 value of 0.3 µg/L for mepanipyrim. Good accuracy and precision values were found when grape juice and wine samples were analysed.

Polymers

A DNA structure, known as triple-stranded DNA, is made up of three oligonucleotide chains that wind around one another to form a triple helix (TFO). Hoogsteen base pairing describes how triple-stranded DNA may be built at certain conditions by the attachment of the third strand to an RNA, PNA, or DNA, which might all be employed as oligonucleotide chains. In each of these situations, the oligonucleotides can be employed as an anchor, in conjunction with a specific bioactive chemical, or as a messenger that enables switching between transcription and replication through the triplex-forming zone. These data are also considered since various illnesses have been linked to the expansion of triplex-prone sequences. In light of metabolic acidosis and associated symptoms, some consideration is given to the impact of several low-molecular-weight compounds, including pH on triplex production in vivo. The review is focused on the development of biomedical oligonucleotides with triplexes.

Chemical Science

A primer that covers the discovery and validation process for novel aptamers and highlights recent applications of aptamers in biosensing and cell separations.

Sensors, 2012

Aptamers are artificial oligonucleotides (DNA or RNA) selected in vitro that bind a broad range of targets with high affinity and specificity; a sensitive yet simple method to utilize aptamers as recognition elements for the development of biosensors (aptasensors) is to transduce the signal electrochemically. So far, aptasensors have been applied to clinical diagnostics and several technologies are in development. Aptasensors will extend the limits of current clinical diagnostics. Although the potential diagnostic applications are unlimited, the most current applications are foreseen in the areas of biomarker detection, cancer clinical testing, detection of infectious microorganisms and viruses. This review attempts to list examples of the research progresses of aptamers in biosensor platforms that have been published in recent years; in particular, we display cases of aptasensors that are already incorporated in clinical diagnostics or have potential applications in clinical diagnostics.

World Mycotoxin Journal, 2011

Mycotoxin analysis and detection in food and drinks is vital for ensuring food quality and safety, eliminating and controlling the risk of consuming contaminated foods, and complying with the legislative limits set by food authorities worldwide. Most analysis of these toxins is still conducted using conventional methods; however, biosensor methods are currently being developed as screening tools for use in field analysis. Biosensors have demonstrated their ability to provide rapid, sensitive, robust and cost-effective quantitative methods for on-site testing. The development of biosensor devices for different mycotoxins has attracted much research interest in recent years with a range of devices being designed and reported in the scientific literature. However, with the advent of nanotechnology and its impact on the evolution of ultrasensitive devices, mycotoxin analysis is also benefiting from the advances taking place in applying nanomaterials in sensors development. This paper re...

Pharmaceuticals, 2018

Cancer and infectious diseases such as Ebola, HIV, tuberculosis, Zika, hepatitis, measles and human schistosomiasis are serious global health hazards. The increasing annual morbidities and mortalities of these diseases have been blamed on drug resistance and the inefficacy of available diagnostic tools, particularly those which are immunologically-based. Antibody-based tools rely solely on antibody production for diagnosis and for this reason they are the major cause of diagnostic delays. Unfortunately, the control of these diseases depends on early detection and administration of effective treatment therefore any diagnostic delay is a huge challenge to curbing these diseases. Hence, there is a need for alternative diagnostic tools, discovery and development of novel therapeutic agents. Studies have demonstrated that aptamers could potentially offer one of the best solutions to these problems. Aptamers are short sequences of either DNA or RNA molecules, which are identified in vitro...

Sensors, 2011

Recent developments in aptamers have led to their widespread use in analytical and diagnostic applications, particularly for biosensing. Previous studies have combined aptamers as ligands with various sensors for numerous applications. However, merging the aptamer developments with guided mode resonance (GMR) devices has not been attempted. This study reports an aptasensor based home built GMR device. The 29-mer thrombin aptamer was immobilized on the surface of a GMR device as a recognizing ligand for thrombin detection. The sensitivity reported in this first trial study is 0.04 nm/μM for thrombin detection in the concentration range from 0.25 to 1 μM and the limit of detection (LOD) is 0.19 μM. Furthermore, the binding affinity constant (K a) measured is in the range of 10 6 M −1. The investigation has demonstrated that such a GMR aptasensor has the required sensitivity for the real time, label-free, in situ detection of thrombin and provides kinetic information related to the binding.

Chemical Communications, 2015

A simple modular aptamer-based sensor (aptasensor) platform was prepared by combining the merits of the rapid and efficient preparation of a self-assembled monolayer of cucurbit[7]uril (CB[7] SAM) and the strong and specific binding affinity of CB[7] to ferrocenemethylammonium (FA), as an ultrastable supramolecular linker, to immobilize aptamers on CB[7] SAM.

Engineering Proceedings, 2021

Optical detection is one of the most used transduction methods in biosensors and apart from the commercially available instruments based on surface plasmon resonance (SPR), an emerging class of devices, based on both silica and plastic optical fibers (POFs), is finding its route. On the other hand, aptamers represent the next-generation biorecognition elements for biosensor implementation, thanks to several characteristics making them more appealing with respect to the conventional antibodies. The joint exploitation of plasmon resonance in plastic optical fibers and aptamers is here reported, focusing the attention on various relevant biological applications (e.g., thrombin, vascular endothelial growth factor (VEGF), and SARS-CoV-2 spike protein).

Pharmaceuticals, 2021

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Sensors, 2020

Selected optical fiber sensors offer extraordinary sensitivity to changes in external refractive (RI), which make them promising for label-free biosensing. In this work the most sensitive ones, namely long-period gratings working at (DTP-LPG) and micro-cavity in-line Mach-Zehnder interferometers (µIMZI) are discussed for application in bacteria sensing. We describe their working principles and RI sensitivity when operating in water environments, which is as high as 20,000 nm/RIU (Refractive index unit) for DTP-LPGs and 27,000 nm/RIU for µIMZIs. Special attention is paid to the methods to enhance the sensitivity by etching and nano-coatings. While the DTP-LPGs offer a greater interaction length and sensitivity to changes taking place at their surface, the µIMZIs are best suited for investigations of sub-nanoliter and picoliter volumes. The capabilities of both the platforms for bacteria sensing are presented and compared for strains of Escherichia coli, lipopolysaccharide E. coli, ou...

Chemical communications (Cambridge, England), 2016

We successfully implemented solvent extraction of short, single-stranded RNA using reverse micelles (water-in-oil microemulsions) with a DNA-surfactant. A thrombin-binding RNA aptamer was enzymatically synthesized and purified by extraction using the reverse micellar system. The extracted RNA aptamer retained thrombin-binding activity after the extraction procedure.

Springer eBooks, 2014

BioMed research international, 2015

Angiogenesis plays a decisive role in the growth and spread of cancer and angiopoietin-2 (Ang2) is in the spotlight of studies for its unique role in modulating angiogenesis. The aim of this study was to introduce a computational simulation approach to screen aptamers with high binding ability for Ang2. We carried out computational simulations of aptamer-protein interactions by using ZDOCK and ZRANK functions in Discovery Studio 3.5 starting from the available information of aptamers generated through the systematic evolution of ligands by exponential enrichment (SELEX) in the literature. From the best of three aptamers on the basis of ZRANK scores, 189 sequences with two-point mutations were created and simulated with Ang2. Then, we used a surface plasmon resonance (SPR) biosensor to test 3 mutant sequences of high ZRANK scores along with a high and a low affinity binding sequence as reported in the literature. We found a selected RNA aptamer has a higher binding affinity and SPR r...

Biosensors

Selective and sensitive detection of human activated protein C (APC) was performed herein by using carbon nanofiber (CNF) and ionic liquid (IL) composite modified pencil graphite electrode (PGE) and electrochemical impedance spectroscopy (EIS) technique. A carbon nanomaterial-based electrochemical aptasensor was designed and implemented for the first time in this study for the solution-phase interaction of DNA-Apt with its cognate protein APC as well as APC inhibitor aptamer–antidote pair. The applicability of this assay developed for the determination of APC in fetal bovine serum (FBS) and its selectivity against different proteins (protein C, thrombin, bovine serum albumin) was also examined. CNF-IL modified aptasensor specific to APC provided the detection limit as 0.23 μg/mL (equal to 3.83 nM) in buffer medium and 0.11 μg/mL (equal to 1.83 nM) in FBS. The duration of the proposed assay from the point of electrode modification to the detection of APC was completed within only 55 ...

IntechOpen eBooks, 2021

Biosensor technology has developed extremely rapidly in recent years. This technology brings along precise measurements as well as specific measurements. Thanks to its ability to be miniaturized and be easily accessible to the end user, it is one-step ahead of other similar methods. The selectivity of biological molecules and the sensitivity of electrochemical methods enable the continuous evolvement of these new technologies. In this chapter, the use of nucleic acids as both recognition agents and target molecules, the way they are used in biosensor technology and their electrical properties are explained in detail with examples. Aptamers, which are synthetic nucleic acids, and their use in electrochemical biosensor systems with different electrochemical and immobilization methods have been compared extensively.

Journal of Materials Chemistry B, 2018

Illustration of the PEC cytosensor fabrication process and its corresponding photocurrents.