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Title: Lab-on-a-Chip Systems for Aptamer-Based Biosensing
Aptamers are oligonucleotides or peptides that are selected from a pool of random sequences that exhibit high affinity toward a specific biomolecular species of interest. Therefore, they are ideal for use as recognition elements and ligands for binding to the target. In recent years, aptamers have gained a great deal of attention in the field of biosensing as the next-generation target receptors that could potentially replace the functions of antibodies. Consequently, it is increasingly becoming popular to integrate aptamers into a variety of sensing platforms to enhance specificity and selectivity in analyte detection. Simultaneously, as the fields of lab-on-a-chip (LOC) technology, point-of-care (POC) diagnostics, and personal medicine become topics of great interest, integration of such aptamer-based sensors with LOC devices are showing promising results as evidenced by the recent growth of literature in this area. The focus of this review article is to highlight the recent progress in aptamer-based biosensor development with emphasis on the integration between aptamers and the various forms of LOC devices including microfluidic chips and paper-based microfluidics. As aptamers are extremely versatile in terms of their utilization in different detection principles, a broad range of techniques are covered including electrochemical, optical, colorimetric, and gravimetric sensing as more » well as surface acoustics waves and transistor-based detection. « less
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National Science Foundation
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  1. Aptamer-immobilized graphene field-effect transistors (GFETs) have become a well-known detection platform in the field of biosensing with various biomarkers such as proteins, bacteria, virus, as well as chemicals. A conventional aptamer immobilization technique on graphene involves a two-step crosslinking process. In the first step, a pyrene derivative is anchored onto the surface of graphene and, in the second step, an amine-terminated aptamer is crosslinked to the pyrene backbone with EDC/NHS (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide) chemistry. However, this process often requires the use of organic solvents such as dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO) which are typically polar aprotic solvents and hence dissolves both polar and nonpolar compounds. The use of such solvents can be especially problematic in the fabrication of lab-on-a-chip or point-of-care diagnostic platforms as they can attack vulnerable materials such as polymers, passivation layers and microfluidic tubing leading to device damage and fluid leakage. To remedy such challenges, in this work, we demonstrate the use of pyrene-tagged DNA aptamers (PTDA) for performing a one-step aptamer immobilization technique to implement a GFET-based biosensor for the detection of Interleukin-6 (IL-6) protein biomarker. In this approach, the aptamer terminal is pre-tagged with a pyrene group which becomes soluble in aqueous solution.more »This obviates the need for using organic solvents, thereby enhancing the device integrity. In addition, an external electric field is applied during the functionalization step to increase the efficiency of aptamer immobilization and hence improved coverage and density. The results from this work could potentially open up new avenues for the use of GFET-based BioMEMS platforms by broadening the choice of materials used for device fabrication and integration.« less
  2. Detection of analytes by means of field-effect transistors bearing ligand-specific receptors is fundamentally limited by the shielding created by the electrical double layer (the “Debye length” limitation). We detected small molecules under physiological high–ionic strength conditions by modifying printed ultrathin metal-oxide field-effect transistor arrays with deoxyribonucleotide aptamers selected to bind their targets adaptively. Target-induced conformational changes of negatively charged aptamer phosphodiester backbones in close proximity to semiconductor channels gated conductance in physiological buffers, resulting in highly sensitive detection. Sensing of charged and electroneutral targets (serotonin, dopamine, glucose, and sphingosine-1-phosphate) was enabled by specifically isolated aptameric stem-loop receptors.

  3. Aptamers have many useful attributes including specific binding to molecular targets. After aptamers are identified, their target binding must be characterized. Fluorescence anisotropy (FA) is one technique that can be used to characterize affinity and to optimize aptamer–target interactions. Efforts to make FA assays more efficient by reducing assay volume and time from mixing to measurement may save time and resources by minimizing consumption of costly reagents. Here, we use thrombin and two thrombin-binding aptamers as a model system to show that plate-based FA experiments can be performed in volumes as low as 2 μL per well with 20 minute incubations with minimal loss in assay precision. We demonstrate that the aptamer–thrombin interaction is best modelled with the Hill equation, indicating cooperative binding. The miniaturization of this assay has implications in drug development, as well as in the efficiency of aptamer selection workflows by allowing for higher throughput aptamer analysis.
  4. Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising for biosensing applications because target-induced conformational change can be directly linked to a functional output. However, traditional evolution methods do not select for the significant conformational change needed to create structure-switching biosensors. Modified selection methods have been described to select for structure-switching architectures, but these remain limited by the need for immobilization. Herein we describe the first homogenous, structure-switching aptamer selection that directly reports on biosensor capacity for the target. We exploit the activity of restriction enzymes to isolate aptamer candidates that undergo target-induced displacement of a short complementary strand. As an initial demonstration of the utility of this approach, we performed selection against kanamycin A. Four enriched candidate sequences were successfully characterized as structure-switching biosensors for detection of kanamycin A. Optimization of biosensor conditions afforded facile detection of kanamycin A (90 μM to 10 mM) with high selectivity over three other aminoglycosides. This research demonstrates a general method to directly select for structure-switching biosensors and can be applied to a broad range of small-molecule targets.
  5. Abstract

    Owing to its great threat to human health and environment, Pb2+pollution has been recognized as a major public problem by the World Health Organization (WHO). Many DNA aptamers have been utilized in the development of Pb2+-detection sensors, but the underlying mechanisms remain elusive. Here, we report three Pb2+-complexed structures of the thrombin binding aptamer (TBA). These high-resolution crystal structures showed that TBA forms intramolecular G-quadruplex and Pb2+is bound by the two G-tetrads in the center. Compared to K+-stabilized G-quadruplexes, the coordinating distance between Pb2+and the G-tetrads are much shorter. The T3T4 and T12T13 linkers play important roles in dimerization and crystallization of TBA, but they are changeable for Pb2+-binding. In combination with mutagenesis and CD spectra, the G8C mutant structure unraveled that the T7G8T9 linker of TBA is also variable. In addition to expansion of the Pb2+-binding aptamer sequences, our study also set up one great example for quick and rational development of other aptamers with similar or optimized binding activity.