Switching the aptamer attachment geometry can dramatically alter the signalling and performance of electrochemical aptamer-based sensors

Chamorro-Garcia, Alejandro; Ortega, Gabriel; Mariottini, Davide; Green, Joshua; Ricci, Francesco; Plaxco, Kevin W.

doi:10.1039/d1cc04557a

Cited by 13 publications

(11 citation statements)

References 33 publications

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“…All surface constructs employed in this work, including 3trunc, included a covalent bond to methylene blue at the 3′ terminus, and one to hexylthiol at the 5′ terminus (see the Methods section for structure). This configuration favors the strongest signaling from E-AB sensors as reported by Chamorro-García and colleagues . Regression analysis of the resulting isotherms revealed an expected destabilization of the folded state of the 3trunc aptamer via a 3-fold worsening of the dissociation constant ( K D = 9.13 ± 1.9 vs 3.92 ± 0.90 μM without functionalization, Figure S1D vs Figure C).…”

Section: Resultsmentioning

confidence: 99%

Optimization of Vancomycin Aptamer Sequence Length Increases the Sensitivity of Electrochemical, Aptamer-Based Sensors In Vivo

et al. 2022

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The measurement of serum vancomycin levels at the clinic is critical to optimizing dosing given the narrow therapeutic window of this antibiotic. Current approaches to quantitate serum vancomycin levels are based on immunoassays, which are multistep methods requiring extensive processing of patient samples. As an alternative, vancomycin-binding electrochemical, aptamer-based sensors (E-ABs) were developed to simplify the workflow of vancomycin monitoring. E-ABs enable the instantaneous measurement of serum vancomycin concentrations without the need for sample dilution or other processing steps. However, the originally reported vancomycin-binding E-ABs had a dissociation constant of 45 μM, which is approximately 1 order of magnitude higher than the recommended trough concentrations of vancomycin measured in patients. This limited sensitivity hinders the ability of E-ABs to accurately support vancomycin monitoring. To overcome this problem, here we sought to optimize the length of the vancomycin-binding aptamer sequence to enable a broader dynamic range in the E-AB platform. Our results demonstrate, via isothermal calorimetry and E-AB calibrations in undiluted serum, that superior affinity and near-equal sensor gain in vitro can be achieved using a one-base-pair-longer aptamer than the truncated sequence originally reported. We validate the impact of the improved binding affinity in vivo by monitoring vancomycin levels in the brain cortex of live mice following intravenous administration. While the original sequence fails to resolve vancomycin concentrations from baseline noise (SNR = 1.03), our newly reported sequence provides an SNR of 1.62 at the same dose.

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Section: Resultsmentioning

confidence: 99%

Optimization of Vancomycin Aptamer Sequence Length Increases the Sensitivity of Electrochemical, Aptamer-Based Sensors In Vivo

et al. 2022

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“…The solid curves are fits to the Hill equation (eqn (3)); the Chi-square values are, for the curves from low to high depletant, 0.004, 0.006, 0.008, 0.007, 0.009 and 0.003. its 5 ′ terminus to the surface of a gold electrode using thiol-ongold self-assembled monolayer chemistry. 7,46,72 Binding induced folding of this receptor alters the rate of electron transfer from the reporter (here methylene blue), producing an easily measurable change when the sensor is interrogated using square wave voltammetry. 8,73 To introduce sequestration to this sensor we added depletant to the NGAL-containing sample in a complex matrix (synthetic urine) prior to measurement.…”

Section: Resultsmentioning

confidence: 99%

“…We fabricated the aptamer based electrochemical sensors (EAB sensors) on gold electrodes, following previously described procedures. 7,45,46 In brief, we cleaned the polycrystalline gold disk electrodes by polishing them with 1 mm monocrystalline diamond suspension on a MicroCloth, sonicated them in ethanol, polished again using this time a 0.05 mm aluminium slurry, and nally sonicated in ethanol. Subsequently, we rinsed the electrodes with double distilled water and cleaned them electrochemically.…”

Section: Electrochemical Sensor Fabricationmentioning

confidence: 99%

The sequestration mechanism as a generalizable approach to improve the sensitivity of biosensors and bioassays

et al. 2022

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“…It was found that aptamer attachment geometry on the electrode could dramatically alter the performance of electrochemical aptasensors. By switching the 3′ and 5′ terminus for electrode binding and reporter tagging, the orientation of aptamers strongly affected the output signals, suggesting that the attachment geometry of aptamers is a worthwhile parameter to optimize in the design of new electrochemical aptasensors [76]. Besides the essential components of electrochemical aptasensors, namely, aptamers, electrode, and electroactive species, the existence of external interference factors in the supporting buffer such as salts, ions, and cell lysates may lead to electrochemical signal errors.…”

Section: Labeled Electrochemical Aptasensorsmentioning

confidence: 99%

Aptasensors for the detection of infectious pathogens: design strategies and point-of-care testing

2022

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The epidemic of infectious diseases caused by contagious pathogens is a life-threatening hazard to the entire human population worldwide. A timely and accurate diagnosis is the critical link in the fight against infectious diseases. Aptamer-based biosensors, the so-called aptasensors, employ nucleic acid aptamers as bio-receptors for the recognition of target pathogens of interest. This review focuses on the design strategies as well as state-of-the-art technologies of aptasensor-based diagnostics for infectious pathogens (mainly bacteria and viruses), covering the utilization of three major signal transducers, the employment of aptamers as recognition moieties, the construction of versatile biosensing platforms (mostly micro and nanomaterial-based), innovated reporting mechanisms, and signal enhancement approaches. Advanced point-of-care testing (POCT) for infectious disease diagnostics are also discussed highlighting some representative ready-to-use devices to address the urgent needs of currently prevalent coronavirus disease 2019 (COVID-19). Pressing issues in aptamer-based technology and some future perspectives of aptasensors are provided for the implementation of aptasensor-based diagnostics into practical application.

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Switching the aptamer attachment geometry can dramatically alter the signalling and performance of electrochemical aptamer-based sensors

Abstract: Electrochemical Aptamer-Based (EAB) sensors, comprised of an electrode bound DNA aptamer with a redox reporter on the distal end, offer the promise of high-frequency, real-time molecular measurements in complex sample...

Cited by 13 publications

References 33 publications

Optimization of Vancomycin Aptamer Sequence Length Increases the Sensitivity of Electrochemical, Aptamer-Based Sensors In Vivo

Optimization of Vancomycin Aptamer Sequence Length Increases the Sensitivity of Electrochemical, Aptamer-Based Sensors In Vivo

The sequestration mechanism as a generalizable approach to improve the sensitivity of biosensors and bioassays

Aptasensors for the detection of infectious pathogens: design strategies and point-of-care testing

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