Hybridization efficiency of molecular beacons bound to gold nanowires: Effect of surface coverage and target length

Kristin B. Cederquist, Christine D. Keating

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Surface-bound nucleic acid probes designed to adopt specific secondary structures are becoming increasingly important in a range of biosensing applications but remain less well characterized than traditional single-stranded probes, which are typically designed to avoid secondary structure. We report the hybridization efficiency for surface-immobilized hairpin DNA probes. Our probes are molecular beacons, carrying a 3′ dye moiety and a 5′ thiol for attachment to gold nanowires, which serve as both scaffolds for probe attachment and quenchers. Hybridization efficiency was dependent on probe surface coverage, reaching a maximum of ∼ 90% at intermediate coverages of (1-2) × 1012 probes/cm2 and dropping to ≤20% at higher or lower coverages. Fluorescence intensity did not track with the number of target molecules bound, and was highest for high probe coverage despite the lower bound targets per square centimeter. Backfilling with short thiolated oligoethylene glycol spacers increased hybridization efficiency at low hairpin probe coverages (∼ (3-4) × 1011 probes/cm2), but not at higher probe coverages (1 × 1012/cm2). We also evaluated the effect of target length by adding up to 50 nonhybridizing nucleotides to the 3′ or 5′ end of the complementary target sequence. Additional nucleotides on the 3′ end of the complementary target sequence (i.e., the end near the nanowire surface) had a much greater impact on hybridization efficiency as compared to nucleotides added to the 5′ end. This work provides guidance in designing sensors in which surface-bound probes designed to adopt secondary structures are used to detect target sequences from solution.

Original languageEnglish (US)
Pages (from-to)18273-18280
Number of pages8
JournalLangmuir
Volume26
Issue number23
DOIs
StatePublished - Dec 7 2010

Fingerprint

beacons
Gold
Nanowires
nanowires
gold
probes
nucleotides
Nucleotides
Nucleic Acid Probes
attachment
Molecular Probes
Glycols
DNA Probes
Sulfhydryl Compounds
Scaffolds
Nucleic acids
nucleic acids
Coloring Agents
Fluorescence
thiols

All Science Journal Classification (ASJC) codes

  • Materials Science(all)
  • Condensed Matter Physics
  • Surfaces and Interfaces
  • Spectroscopy
  • Electrochemistry

Cite this

@article{4a698f55ab8a4e4cb8d4a915fd9bc288,
title = "Hybridization efficiency of molecular beacons bound to gold nanowires: Effect of surface coverage and target length",
abstract = "Surface-bound nucleic acid probes designed to adopt specific secondary structures are becoming increasingly important in a range of biosensing applications but remain less well characterized than traditional single-stranded probes, which are typically designed to avoid secondary structure. We report the hybridization efficiency for surface-immobilized hairpin DNA probes. Our probes are molecular beacons, carrying a 3′ dye moiety and a 5′ thiol for attachment to gold nanowires, which serve as both scaffolds for probe attachment and quenchers. Hybridization efficiency was dependent on probe surface coverage, reaching a maximum of ∼ 90{\%} at intermediate coverages of (1-2) × 1012 probes/cm2 and dropping to ≤20{\%} at higher or lower coverages. Fluorescence intensity did not track with the number of target molecules bound, and was highest for high probe coverage despite the lower bound targets per square centimeter. Backfilling with short thiolated oligoethylene glycol spacers increased hybridization efficiency at low hairpin probe coverages (∼ (3-4) × 1011 probes/cm2), but not at higher probe coverages (1 × 1012/cm2). We also evaluated the effect of target length by adding up to 50 nonhybridizing nucleotides to the 3′ or 5′ end of the complementary target sequence. Additional nucleotides on the 3′ end of the complementary target sequence (i.e., the end near the nanowire surface) had a much greater impact on hybridization efficiency as compared to nucleotides added to the 5′ end. This work provides guidance in designing sensors in which surface-bound probes designed to adopt secondary structures are used to detect target sequences from solution.",
author = "Cederquist, {Kristin B.} and Keating, {Christine D.}",
year = "2010",
month = "12",
day = "7",
doi = "10.1021/la1031703",
language = "English (US)",
volume = "26",
pages = "18273--18280",
journal = "Langmuir",
issn = "0743-7463",
publisher = "American Chemical Society",
number = "23",

}

Hybridization efficiency of molecular beacons bound to gold nanowires : Effect of surface coverage and target length. / Cederquist, Kristin B.; Keating, Christine D.

In: Langmuir, Vol. 26, No. 23, 07.12.2010, p. 18273-18280.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Hybridization efficiency of molecular beacons bound to gold nanowires

T2 - Effect of surface coverage and target length

AU - Cederquist, Kristin B.

AU - Keating, Christine D.

PY - 2010/12/7

Y1 - 2010/12/7

N2 - Surface-bound nucleic acid probes designed to adopt specific secondary structures are becoming increasingly important in a range of biosensing applications but remain less well characterized than traditional single-stranded probes, which are typically designed to avoid secondary structure. We report the hybridization efficiency for surface-immobilized hairpin DNA probes. Our probes are molecular beacons, carrying a 3′ dye moiety and a 5′ thiol for attachment to gold nanowires, which serve as both scaffolds for probe attachment and quenchers. Hybridization efficiency was dependent on probe surface coverage, reaching a maximum of ∼ 90% at intermediate coverages of (1-2) × 1012 probes/cm2 and dropping to ≤20% at higher or lower coverages. Fluorescence intensity did not track with the number of target molecules bound, and was highest for high probe coverage despite the lower bound targets per square centimeter. Backfilling with short thiolated oligoethylene glycol spacers increased hybridization efficiency at low hairpin probe coverages (∼ (3-4) × 1011 probes/cm2), but not at higher probe coverages (1 × 1012/cm2). We also evaluated the effect of target length by adding up to 50 nonhybridizing nucleotides to the 3′ or 5′ end of the complementary target sequence. Additional nucleotides on the 3′ end of the complementary target sequence (i.e., the end near the nanowire surface) had a much greater impact on hybridization efficiency as compared to nucleotides added to the 5′ end. This work provides guidance in designing sensors in which surface-bound probes designed to adopt secondary structures are used to detect target sequences from solution.

AB - Surface-bound nucleic acid probes designed to adopt specific secondary structures are becoming increasingly important in a range of biosensing applications but remain less well characterized than traditional single-stranded probes, which are typically designed to avoid secondary structure. We report the hybridization efficiency for surface-immobilized hairpin DNA probes. Our probes are molecular beacons, carrying a 3′ dye moiety and a 5′ thiol for attachment to gold nanowires, which serve as both scaffolds for probe attachment and quenchers. Hybridization efficiency was dependent on probe surface coverage, reaching a maximum of ∼ 90% at intermediate coverages of (1-2) × 1012 probes/cm2 and dropping to ≤20% at higher or lower coverages. Fluorescence intensity did not track with the number of target molecules bound, and was highest for high probe coverage despite the lower bound targets per square centimeter. Backfilling with short thiolated oligoethylene glycol spacers increased hybridization efficiency at low hairpin probe coverages (∼ (3-4) × 1011 probes/cm2), but not at higher probe coverages (1 × 1012/cm2). We also evaluated the effect of target length by adding up to 50 nonhybridizing nucleotides to the 3′ or 5′ end of the complementary target sequence. Additional nucleotides on the 3′ end of the complementary target sequence (i.e., the end near the nanowire surface) had a much greater impact on hybridization efficiency as compared to nucleotides added to the 5′ end. This work provides guidance in designing sensors in which surface-bound probes designed to adopt secondary structures are used to detect target sequences from solution.

UR - http://www.scopus.com/inward/record.url?scp=78650247157&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=78650247157&partnerID=8YFLogxK

U2 - 10.1021/la1031703

DO - 10.1021/la1031703

M3 - Article

C2 - 21038880

AN - SCOPUS:78650247157

VL - 26

SP - 18273

EP - 18280

JO - Langmuir

JF - Langmuir

SN - 0743-7463

IS - 23

ER -