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Label-free sensing of adenosine based on force variations induced by molecular recognition.

Li J, Li Q, Ciacchi LC, Wei G - Biosensors (Basel) (2015)

Bottom Line: The sensitive force response to molecular recognition provided an adenosine detection limit in the range of 0.1 to 1 nM.The addition of guanosine, cytidine, and uridine had no significant interference with the sensing of adenosine, indicating a strong selectivity of this sensor architecture.In addition, operational parameters that may affect the sensor, such as loading rate and solution ionic strength, were investigated.

View Article: PubMed Central - PubMed

Affiliation: Hybrid Materials Interfaces Group, Faculty of Production Engineering, University of Bremen, Am Fallturm 1, 28359 Bremen, Germany. jinfeng@uni-bremen.de.

ABSTRACT
We demonstrate a simple force-based label-free strategy for the highly sensitive sensing of adenosine. An adenosine ssDNA aptamer was bound onto an atomic force microscopy (AFM) probe by covalent modification, and the molecular-interface adsorption force between the aptamer and a flat graphite surface was measured by single-molecule force spectroscopy (SMFS). In the presence of adenosine, the molecular recognition between adenosine and the aptamer resulted in the formation of a folded, hairpin-like DNA structure and hence caused a variation of the adsorption force at the graphite/water interface. The sensitive force response to molecular recognition provided an adenosine detection limit in the range of 0.1 to 1 nM. The addition of guanosine, cytidine, and uridine had no significant interference with the sensing of adenosine, indicating a strong selectivity of this sensor architecture. In addition, operational parameters that may affect the sensor, such as loading rate and solution ionic strength, were investigated.

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Modification of the AFM probes and conjugation of the ssDNA aptamer.
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biosensors-05-00085-f001: Modification of the AFM probes and conjugation of the ssDNA aptamer.

Mentions: Figure 1 shows the procedure used for the modification of AFM probes. Before modification, all AFM probes were immersed in newly prepared Piranha solution (7/3 v/v, 98% H2SO4, 30% H2O2) for 30 min to remove possible organic contaminates. Caution: the piranha solution has a very strong oxidizing power and is extremely dangerous to handle. Goggles, face shields, and gloves are needed for protection. Then the probes were rinsed with large amount of ultrapure water and ethanol (99%) several times. The probes were then silanized by a mixed solution of 3-aminopropyl triethoxysilane (APTES) and triethoxy(ethyl)silane (TEES) (1% in toluene, 1/4 v/v, APTES/TEES) to functionalize their surfaces with amino groups. In this step, instead of immersing the whole probes into the mixed solution, they were hung vertically by tweezers over the solution and adjusted to submerge into the solution only a small part of the probe. This technique effectively reduces the unwanted functionalization of parts of the probes other than the tip, hence reducing the total amount of DNA aptamer linked to the probe and eventually reducing the adenosine detection limit. After 20 min immersion, the probes were rinsed with ethanol and ultrapure water. They were then transferred into 4,7,10,13,16,19,22,25,32,35,38,41,44,47,50,53-Hexadecaoxa-28,29-dithiahexapentacontanedioic acid di-N-succinimidyl ester (PEG-NHS ester disulfide (n = 7)) (0.1 mg/mL, 100 μL) for 1 h to bind the PEG-NHS ester disulfide to the AFM probes via covalent interaction between surface-bound NH2 groups and the NHS ester groups. The probes were subsequently rinsed with ultrapure water and immersed into the ssDNA aptamer solution (100 nM, 100 μL) for 30 min to link the aptamer to the probes. Finally, the aptamer-modified AFM probes were rinsed with a large amount of ultrapure water to remove non-covalently adsorbed DNA molecules prior to the SMFS experiments.


Label-free sensing of adenosine based on force variations induced by molecular recognition.

Li J, Li Q, Ciacchi LC, Wei G - Biosensors (Basel) (2015)

Modification of the AFM probes and conjugation of the ssDNA aptamer.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384084&req=5

biosensors-05-00085-f001: Modification of the AFM probes and conjugation of the ssDNA aptamer.
Mentions: Figure 1 shows the procedure used for the modification of AFM probes. Before modification, all AFM probes were immersed in newly prepared Piranha solution (7/3 v/v, 98% H2SO4, 30% H2O2) for 30 min to remove possible organic contaminates. Caution: the piranha solution has a very strong oxidizing power and is extremely dangerous to handle. Goggles, face shields, and gloves are needed for protection. Then the probes were rinsed with large amount of ultrapure water and ethanol (99%) several times. The probes were then silanized by a mixed solution of 3-aminopropyl triethoxysilane (APTES) and triethoxy(ethyl)silane (TEES) (1% in toluene, 1/4 v/v, APTES/TEES) to functionalize their surfaces with amino groups. In this step, instead of immersing the whole probes into the mixed solution, they were hung vertically by tweezers over the solution and adjusted to submerge into the solution only a small part of the probe. This technique effectively reduces the unwanted functionalization of parts of the probes other than the tip, hence reducing the total amount of DNA aptamer linked to the probe and eventually reducing the adenosine detection limit. After 20 min immersion, the probes were rinsed with ethanol and ultrapure water. They were then transferred into 4,7,10,13,16,19,22,25,32,35,38,41,44,47,50,53-Hexadecaoxa-28,29-dithiahexapentacontanedioic acid di-N-succinimidyl ester (PEG-NHS ester disulfide (n = 7)) (0.1 mg/mL, 100 μL) for 1 h to bind the PEG-NHS ester disulfide to the AFM probes via covalent interaction between surface-bound NH2 groups and the NHS ester groups. The probes were subsequently rinsed with ultrapure water and immersed into the ssDNA aptamer solution (100 nM, 100 μL) for 30 min to link the aptamer to the probes. Finally, the aptamer-modified AFM probes were rinsed with a large amount of ultrapure water to remove non-covalently adsorbed DNA molecules prior to the SMFS experiments.

Bottom Line: The sensitive force response to molecular recognition provided an adenosine detection limit in the range of 0.1 to 1 nM.The addition of guanosine, cytidine, and uridine had no significant interference with the sensing of adenosine, indicating a strong selectivity of this sensor architecture.In addition, operational parameters that may affect the sensor, such as loading rate and solution ionic strength, were investigated.

View Article: PubMed Central - PubMed

Affiliation: Hybrid Materials Interfaces Group, Faculty of Production Engineering, University of Bremen, Am Fallturm 1, 28359 Bremen, Germany. jinfeng@uni-bremen.de.

ABSTRACT
We demonstrate a simple force-based label-free strategy for the highly sensitive sensing of adenosine. An adenosine ssDNA aptamer was bound onto an atomic force microscopy (AFM) probe by covalent modification, and the molecular-interface adsorption force between the aptamer and a flat graphite surface was measured by single-molecule force spectroscopy (SMFS). In the presence of adenosine, the molecular recognition between adenosine and the aptamer resulted in the formation of a folded, hairpin-like DNA structure and hence caused a variation of the adsorption force at the graphite/water interface. The sensitive force response to molecular recognition provided an adenosine detection limit in the range of 0.1 to 1 nM. The addition of guanosine, cytidine, and uridine had no significant interference with the sensing of adenosine, indicating a strong selectivity of this sensor architecture. In addition, operational parameters that may affect the sensor, such as loading rate and solution ionic strength, were investigated.

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