Aptasensors design considerations

With the advancement of biotechnology, aptamer-based sensors have received intense attention in both research and commercial worlds. This is driven by the advantages of small molecule size, chemical stability, and cost effectiveness of aptamers over the conventional analyte detection using antibodies. This paper explores the aptasensors from a designer perspective and discusses the aptasensor design considerations by giving an overview of surface functionalization techniques and the existing mechanisms used to detect biomolecular interactions. It also expounds the factors that influence the accuracy and sensitivity of aptasensors.

Aptasensors : a review

Aptamers are a promising class of agents for biomolecules detection due to their small size, chemical stability and cost effectiveness over conventional bioreceptors such as antibodies. Recent advances in micro/nano-fabrication and biotechnology have driven active participation of engineers and molecular biologists in the development of aptasensors. This review examines aptasensors from a developer standpoint discussing surface immobilization techniques and mechanisms used to detect biomolecular interactions in the context of biotechnology and nanomedicine. The factors that affect accuracy, sensitivity and stability of aptasensors are also addressed.

Chimeric aptamers in cancer cell-targeted drug delivery

Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets ("apatopes") with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptameraptamer, aptamernonaptamer biomacromolecules (siRNAs, proteins) and aptamernanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe3O4) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities.

Surface-stress-based microcantilever aptasensor

This thesis presents the design, modelling, fabrication, and biological evaluation of a microcantilever-based aptasensor. It is the first reported work on aptasensors with aptamer immobilized on a bare SU-8 surface. Aptasensor surface funtionalisation was achieved using gas plasma treatment. Label-free detection of thrombin molecules using the aptasensor was successfully demonstrated.

The use of sensitive chemical antibodies for diagnosis: detection of low levels of Epcam in breast cancer

EpCAM is expressed at low levels in a variety of normal human epithelial tissues, but is overexpressed in 70–90% of carcinomas. From a clinico-pathological point of view, this has both prognostic and therapeutic significance. EpCAM was first suggested as a therapeutic target for the treatment of epithelial cancers in the 1990s. However, following several immunotherapy trials, the results have been mixed. It has been suggested that this is due, at least in part, to an unknown level of EpCAM expression in the tumors being targeted. Thus, selection of patients who would benefit from EpCAM immunotherapy by determining EpCAM status in the tumor biopsies is currently undergoing vigorous evaluation. However, current EpCAM antibodies are not robust enough to be able to detect EpCAM expression in all pathological tissues.

Here we report a newly developed EpCAM RNA aptamer, also known as a chemical antibody, which is not only specific but also more sensitive than current antibodies for the detection of EpCAM in formalin-fixed paraffin-embedded primary breast cancers. This new aptamer, together with our previously described aptamer, showed no non- specific staining or cross-reactivity with tissues that do not express EpCAM. They were able to reliably detect target proteins in breast cancer xenograft where an anti-EpCAM antibody (323/A3) showed limited or no reactivity. Our results demonstrated a more robust detection of EpCAM using RNA aptamers over antibodies in clinical samples with chromogenic staining. This shows the potential of aptamers in the future of histopathological diagnosis and as a tool to guide targeted immunotherapy.

Aptamers as theranostic agents: modifications, serum stability and functionalisation

Aptamers, and the selection process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) used to generate them, were first described more than twenty years ago. Since then, there have been numerous modifications to the selectionprocedures. This review discusses the use of modified bases as a means of enhancing serum stability and producing effective therapeutic tools, as well as functionalising these nucleic acids to be used as potential diagnostic agents.

Selection of DNA aptamers against epithelial cell adhesion molecule for cancer cell imaging and circulating tumor cell capture

Epithelial cell adhesion molecule (EpCAM) is overexpressed in most solid cancers and is an ideal antigen for clinical applications in cancer diagnosis, prognosis, imaging, and therapy. Currently, most of the EpCAM-based diagnostic, prognostic, and therapeutic strategies rely on the anti-EpCAM antibody. However, the use of EpCAM antibody is restricted due to its large size and instability. In this study, we have successfully identified DNA aptamers that selectively bind human recombinant EpCAM protein. The aptamers can specifically recognize a number of live human cancer cells derived from breast, colorectal, and gastric cancers that express EpCAM but not bind to EpCAM-negative cells. Among the aptamer sequences identified, a hairpin-structured sequence SYL3 was optimized in length, resulting in aptamer sequence SYL3C. The Kd values of the SYL3C aptamer against breast cancer cell line MDA-MB-231 and gastric cancer cell line Kato III were found to be 38±9 and 67±8 nM, respectively, which are better than that of the full-length SYL3 aptamer. Flow cytometry analysis results indicated that the SYL3C aptamer was able to recognize target cancer cells from mixed cells in cell media. When used to capture cancer cells, up to 63% cancer cell capture efficiency was achieved with about 80% purity. With the advantages of small size, easy synthesis, good stability, high binding affinity, and selectivity, the DNA aptamers reported here against cancer biomarker EpCAM will facilitate the development of novel targeted cancer therapy, cancer cell imaging, and circulating tumor cell detection.

A surface-stress-based microcantilever aptasensor

Biosensors based on microcantilevers convert biological recognition events into measurable mechanical displacements. They offer advantages such as small size, low sample volume, label-free detection, ease of integration, high-throughput analysis, and low development cost. The design and development of a microcantilever-based aptasensor employing SU-8 polymer as the fabrication material is presented in this paper. Aptamers are employed as bioreceptor elements because they exhibit superior specificity compared to antibodies due to their small size and physicochemical stability. To immobilise thrombin DNA aptamer on the bare SU-8 surface of the aptasensor, a combined plasma mode treatment method is implemented which modifies the surface of the aptasensor. Label-free detection of thrombin molecules using the fabricated aptasensor is successfully demonstrated. The measured deflection is one order of magnitude higher than that of a silicon nitride microcantilever biosensor. The developed aptasensor also demonstrates high specificity.

A surface-stress-based microcantilever aptasensor

Biosensors based on microcantilevers convert biological recognition events into measurable mechanical displacements. They offer advantages such as small size, low sample volume, label-free detection, ease of integration, high-throughput analysis, and low development cost. The design and development of a microcantilever-based aptasensor employing SU-8 polymer as the fabrication material is presented in this paper. Aptamers are employed as bioreceptor elements because they exhibit superior specificity compared to antibodies due to their small size and physicochemical stability. To immobilise thrombin DNA aptamer on the bare SU-8 surface of the aptasensor, a combined plasma mode treatment method is implemented which modifies the surface of the aptasensor. Label-free detection of thrombin molecules using the fabricated aptasensor is successfully demonstrated. The measured deflection is one order of magnitude higher than that of a silicon nitride microcantilever biosensor. The developed aptasensor also demonstrates high specificity.

Theoretical modeling and experimental validation of surface stress in thrombin aptasensor

Adsorption of target molecules on the immobilized microcantilever surface produced beam displacement due to the differential surface stress generated between the immobilized and non-immobilized surface. Surface stress is caused by the intermolecular forces between the molecules. Van der Waals, electrostatic forces, hydrogen bonding, hydrophobic effect and steric hindrance are some of the intermolecular forces involved. A theoretical framework describing the adsorption-induced microcantilever displacement is derived in this paper. Experimental displacement of thrombin aptamer-thrombin interactions was carried out. The relation between the electrostatic interactions involved between adsorbates (thrombin) as well as adsorbates and substrates (thrombin aptamer) and the microcantilever beam displacement utilizing the proposed mathematical model was quantified and compared to the experimental value. This exercise is important to aid the designers in microcantilever sensing performance optimization.

Molecular insights and oligonucleotide based targeted gene therapy against cancer

The thesis helps to unravel the function of T lymphoma invasion and metastasis protein (TIAM1) and nucleolin, a nucleolar protein in retinoblastoma tumorigenesis. Aptamer based targeted imaging; drug and gene delivery to retinoblastoma and epithelial cancer cells was attained. The work work finally opened up avenues for cancer stem cell targeting using aptamers, imaging of cancer cells using novel bio-orthogonal agent and use of aptamer for blocking the miRNA-17-92 cluster maturation.

Nucleic acid-based aptamers: applications, development and clinical trials

Short single-stranded oligonucleotides called aptamers, often termed as chemical antibodies, have been developed as powerful alternatives to traditional antibodies with respect to their obvious advantages like high specificity and affinity, longer shelf-life, easier manufacturing protocol, freedom to introduce chemical modifications for further improvement, etc. Reiterative selection process of aptamers over 10-15 cycles starting from a large initial pool of random nucleotide sequences renders them with high binding affinity, thereby making them extremely specific for their targets. Aptamer-based detection systems are well investigated and likely to displace primitive detection systems. Aptamer chimeras (combination of aptamers with another aptamer or biomacromolecule or chemical moiety) have the potential activity of both the parent molecules, and thus hold the capability to perform diverse functions at the same time. Owing to their extremely high specificity and lack of immunogenicity or pathogenicity, a number of other aptamers have recently entered clinical trials and have garnered favorable attention from pharmaceutical companies. Promising results from the clinical trials provide new hope to change the conventional style of therapy. Aptamers have attained high therapeutic relevance in a short time as compared to synthetic drugs and/or other modes of therapy. This review follows the various trends in aptamer technology including production, selection, modifications and success in clinical fields. It focusses largely on the various applications of aptamers which mainly depend upon their selection procedures. The review also sheds light on various modifications and chimerizations that have been implemented in order to improve the stability and functioning of the aptamers, including introduction of locked nucleic acids (LNAs). The application of various aptamers in detection systems has been discussed elaborately in order to stress on their role as efficient diagnostic agents. The key aspect of this review is focused on success of aptamers on the basis of their performance in clinical trials for various diseases.

Targeting VEGF with LNA-stabilized G-rich oligonucleotide for efficient breast cancer inhibition

In this study, we investigated the efficacy of an LNA (locked nucleic acid)-modified DNA aptamer named RNV66 targeting VEGF against various breast cancer cell lines. Our results demonstrate that RNV66 efficiently inhibits breast cancer cell proliferation both in vitro and in vivo. Introduction of LNA nucleotides were crucial for higher efficacy. Furthermore, the binding interaction of RNV66 with VEGF was investigated using molecular dynamic simulations leading to the first computational model of the LNA aptamer-VEGF complex blocking its interaction with VEGF-receptor.

Quick chip assay using locked nucleic acid modified epithelial cell adhesion molecule and nucleolin aptamers for the capture of circulating tumor cells

The role of circulating tumor cells (CTCs) in disease diagnosis, prognosis, monitoring of the therapeutic efficacy, and clinical decision making is immense and has attracted tremendous focus in the last decade. We designed and fabricated simple, flat channel microfluidic devices polydimethylsiloxane (PDMS based) functionalized with locked nucleic acid (LNA) modified aptamers (targeting epithelial cell adhesion molecule (EpCAM) and nucleolin expression) for quick and efficient capture of CTCs and cancer cells. With optimized flow rates (10 μl/min), it was revealed that the aptamer modified devices offered reusability for up to six times while retaining optimal capture efficiency (>90%) and specificity. High capture sensitivity (92%) and specificity (100%) was observed in whole blood samples spiked with Caco-2 cells (10-100 cells/ml). Analysis of blood samples obtained from 25 head and neck cancer patients on the EpCAM LNA aptamer functionalized chip revealed that an average count of 5 ± 3 CTCs/ml of blood were captured from 22/25 samples (88%). EpCAM intracellular domain (EpICD) immunohistochemistry on 9 oral squamous cell carcinomas showed the EpICD positivity in the tumor cells, confirming the EpCAM expression in CTCs from head and neck cancers. These microfluidic devices also maintained viability for in vitro culture and characterization. Use of LNA modified aptamers provided added benefits in terms of cost effectiveness due to increased reusability and sustainability of the devices. Our results present a robust, quick, and efficient CTC capture platform with the use of simple PDMS based devices that are easy to fabricate at low cost and have an immense potential in cancer diagnosis, prognosis, and therapeutic planning.