Aptamers are short oligonucleotides isolated in vitro from randomized libraries that can bind to specific molecules with high affinity, and offer a number of advantages relative to antibodies as biorecognition elements in biosensors. However, it remains difficult and labor‐intensive to develop aptamer‐based sensors for small‐molecule detection. Here, we review the challenges and advances in the isolation and characterization of small‐molecule‐binding DNA aptamers and their use in sensors. First, we discuss in vitro methodologies for the isolation of aptamers, and provide guidance on selecting the appropriate strategy for generating aptamers with optimal binding properties for a given application. We next examine techniques for characterizing aptamer–target binding and structure. Afterwards, we discuss various small‐molecule sensing platforms based on original or engineered aptamers, and their detection applications. Finally, we conclude with a general workflow to develop aptamer‐based small‐molecule sensors for real‐world applications.
A self-assembling split aptamer multiplex assay for SARS-COVID19 and miniaturization of a malachite green DNA-based aptamer
Multiplex assays often rely on expensive sensors incorporating covalently linked fluorescent dyes. Herein, we
developed a self-assembling aptamer-based multiplex assay. This multiplex approach utilizes a previously
established split aptamer sensor in conjugation with a novel split aptamer sensor based upon a malachite green
DNA aptamer. This system was capable of simultaneous fluorescent detection of two SARS COVID-19-related
sequences in one sample with individual sensors that possesses a limit of detection (LOD) in the low nM
range. Optimization of the Split Malachite Green (SMG) sensor yielded a minimized aptamer construct, Mini-MG,
capable of inducing fluorescence of malachite green in both a DNA hairpin and sensor format.
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- Award ID(s):
- 1907824
- PAR ID:
- 10487984
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Sensors and Actuators Reports
- ISSN:
- 2666-0539
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Aptamers are short oligonucleotides isolated in vitro from randomized libraries that can bind to specific molecules with high affinity, and offer a number of advantages relative to antibodies as biorecognition elements in biosensors. However, it remains difficult and labor‐intensive to develop aptamer‐based sensors for small‐molecule detection. Here, we review the challenges and advances in the isolation and characterization of small‐molecule‐binding DNA aptamers and their use in sensors. First, we discuss in vitro methodologies for the isolation of aptamers, and provide guidance on selecting the appropriate strategy for generating aptamers with optimal binding properties for a given application. We next examine techniques for characterizing aptamer–target binding and structure. Afterwards, we discuss various small‐molecule sensing platforms based on original or engineered aptamers, and their detection applications. Finally, we conclude with a general workflow to develop aptamer‐based small‐molecule sensors for real‐world applications.
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Neuropeptide Y (NPY) plays a central role in a variety of emotional and physiological functions in humans, such as forming a part of the body′s response to stress and anxiety. This work compares the impact of MCH and PEG spacer molecules on the performance of a potentiometric NPY sensor. An NPY‐specific DNA aptamer with thiol termination was immobilized onto a gold electrode surface. The performance of the sensor is compared when either an MCH‐ or PEG‐based self‐assembled monolayer is formed following aptamer immobilization. Backfilling the surface with alkanethiol spacer molecules like these is key for proper conformational folding of aptamer‐target binding. Non‐specific adhesion of NPY to the MCH‐based sensor surface was observed via surface plasmon resonance (SPR), and then confirmed via potentiometry. It is then shown that PEG improves the sensor′s sensitivity to NPY compared to the surfaces with an MCH‐based SAM. We achieve the detection of picomolar range NPY levels in buffer with a sensitivity of 36.1 mV/decade for the aptamer and PEG‐based sensor surface, thus demonstrating the promise of potentiometric sensing of NPY for future wearable deployment. The sensor′s selectivity was also studied via exposure to cortisol, a different stress marker, resulting in a 13x smaller differential voltage (aptamer‐specific) response compared to that of NPY.more » « less
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null (Ed.)We report the effectiveness of silver nanocluster (Ag-NC) against the biofilm of Pseudomonas aeruginosa (PA). Two DNA aptamers specific for PA and part of their sequences were chosen as templates for growing the Ag-NC. While circular dichroism (CD) studies determined the presence of secondary structures, UV/Vis absorption, and fluorescence spectroscopic studies confirmed the formation of the fluorescent Ag-NC on the DNA templates. Furthermore, mesoscopic physics-based partial wave spectroscopy (PWS) was used to analyze the backscattered light signal that can detect the degree of nanoscale mass density/refractive index fluctuations to identify the biofilm formation, comparatively among the different aptamers with respect to the control sample. The importance of the secondary structure of the aptamer DNA in targeting, successfully binding with the cells and delivering the Ag-NC, is evidenced by the decrease in disorder strength (Ld) of the Ag-NC treated samples compared to the untreated PA cells, which showed the abundance of higher Ld in the PWS studies. The higher Ld value attributed to the higher mass density fluctuations and the formation of biofilm. We envision this study to open a new avenue in using a powerful optical microscopic technique like PWS in detection, and DNA aptamer enclosed silver nanoclusters to prevent biofilms for opportunist pathogens like Pseudomonas aeruginosa.more » « less
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null (Ed.)We report the effectiveness of silver nanocluster (Ag-NC) against the biofilm of Pseudomonas aeruginosa (PA). Two DNA aptamers specific for PA and part of their sequences were chosen as templates for growing the Ag-NC. While circular dichroism (CD) studies determined the presence of secondary structures, UV/Vis absorption, and fluorescence spectroscopic studies confirmed the formation of the fluorescent Ag-NC on the DNA templates. Furthermore, mesoscopic physics-based partial wave spectroscopy (PWS) was used to analyze the backscattered light signal that can detect the degree of nanoscale mass density/refractive index fluctuations to identify the biofilm formation, comparatively among the different aptamers with respect to the control sample. The importance of the secondary structure of the aptamer DNA in targeting, successfully binding with the cells and delivering the Ag-NC, is evidenced by the decrease in disorder strength (Ld) of the Ag-NC treated samples compared to the untreated PA cells, which showed the abundance of higher Ld in the PWS studies. The higher Ld value attributed to the higher mass density fluctuations and the formation of biofilm. We envision this study to open a new avenue in using a powerful optical microscopic technique like PWS in detection, and DNA aptamer enclosed silver nanoclusters to prevent biofilms for opportunist pathogens like Pseudomonas aeruginosa.more » « less
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