SPAD aptasensor for the detection of circulating protein biomarkers.

Biosensors & bioelectronics (2015-01-31)
Laura Pasquardini, Lucio Pancheri, Cristina Potrich, Alessandro Ferri, Claudio Piemonte, Lorenzo Lunelli, Lucia Napione, Valentina Comunanza, Maria Alvaro, Lia Vanzetti, Federico Bussolino, Cecilia Pederzolli
ABSTRACT

The need for decentralized clinical tests together with the concept of time and cost saving are pushing the development of portable, miniaturized, compact biosensors with diagnostic and prognostic purpose. Here, we propose an innovative detection system based on a Single Photon Avalanche Diode (SPAD) with high sensitivity and low noise, crucial features for an efficient chemiluminescence biosensor. The SPAD detector, having 60 µm diameter, has a Photon Detection Efficiency higher than 55% at 425 nm and a Dark Count Rate lower than 100 Hz at room temperature. Our design allows a good optical coupling efficiency between sample and detector. A specific biofunctional surface was implemented taking advantage of aptamers, short DNA sequences having high selectivity and affinity toward their targets. We successfully detected physiological levels of Vascular Endothelial Growth Factor (VEGF), a circulating protein biomarker highly correlated with cancer. The SPAD aptasensor showed a Limit of Detection (LoD) in the pM range, stability (up to 42 days) and re-usability (up to seven cycles). This compact biosensor is therefore a promising step toward the actual use of portable microdevices in diagnostics.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Hydrochloric acid, ACS reagent, 37%
Sigma-Aldrich
Toluene, anhydrous, 99.8%
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Hydrochloric acid, ACS reagent, 37%
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Sodium hydroxide, reagent grade, ≥98%, pellets (anhydrous)
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Glycine, suitable for electrophoresis, ≥99%
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Glycine, ReagentPlus®, ≥99% (HPLC)
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Sodium hydroxide solution, 50% in H2O
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Sodium hydroxide, ACS reagent, ≥97.0%, pellets
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Urea, ACS reagent, 99.0-100.5%
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Toluene, ACS reagent, ≥99.5%
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Urea, powder, BioReagent, for molecular biology, suitable for cell culture
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Sodium hydroxide solution, BioUltra, for molecular biology, 10 M in H2O
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Sodium hydroxide, BioXtra, ≥98% (acidimetric), pellets (anhydrous)
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Hydrogen chloride solution, 4.0 M in dioxane
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Hydrochloric acid solution, 1.0 N, BioReagent, suitable for cell culture
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Sodium hydroxide solution, 1.0 N, BioReagent, suitable for cell culture
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Hydrochloric acid, 36.5-38.0%, BioReagent, for molecular biology
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Hydrochloric acid, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., fuming, ≥37%, APHA: ≤10
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(3-Mercaptopropyl)trimethoxysilane, 95%
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Toluene, suitable for HPLC, 99.9%
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Glycine, BioUltra, for molecular biology, ≥99.0% (NT)
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Urea, ReagentPlus®, ≥99.5%, pellets
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Hydrochloric acid solution, volumetric, 0.1 M HCl (0.1N), endotoxin free
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Sodium hydroxide concentrate, 0.1 M NaOH in water (0.1N), Eluent concentrate for IC
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Sodium hydroxide, puriss., meets analytical specification of Ph. Eur., BP, NF, E524, 98-100.5%, pellets
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Glycine, ACS reagent, ≥98.5%
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Hydrogen chloride solution, 2.0 M in diethyl ether
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Sodium hydroxide solution, 49-51% in water, eluent for IC
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Glycine, from non-animal source, meets EP, JP, USP testing specifications, suitable for cell culture, ≥98.5%
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Urea, 8 M (after reconstitution with 16 mL high purity water)