Detection of a K-ras point mutation employing peptide nucleic acid at the surface of a SPR biosensor

Yasunobu Sato, Keiji Fujimoto, Haruma Kawaguchi

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

A fundamental study of the usage of peptide nucleic acid (PNA) and DNA at the surface of a surface plasmon resonance (SPR) biosensor for detecting a specific point mutation in the K-ras gene for early detection of cancers is reported. The extents of hybridization using DNA analytes of different length which are targets were studied at 25 and 40°C. The 15mer PNA ligand immobilized to the surface of the sensor had a high ability to detect a 11mer DNA analyte point mutation at 25 and 40°C. On the contrary, when DNA was used as a ligand, detection was only confirmed at 40°C which indicates that the difference of melting temperature (Tm) between the complementary and mismatched analyte was a key factor for point mutation detection. This also implied the decrease of Tm by immobilizing a probe to a surface since it was below the one in solution. Therefore, effects from the surface are seen in this system and the surrounding environment makes a difference to the discrimination process. To understand the process in detail, kinetic analysis was performed and association constants were found to be larger for DNA ligands implying the effect coming from the sensor surface. In this case negative charges of the carboxyl groups of the matrix had a tendency to repel negatively charged DNA ligands. Affinity constants (KA) were calculated and as expected, the ratio of KA between the complementary and mismatched analyte was of the order of 102 for PNA interacting with 11mer analytes, making it a promising probe for genetic diagnosis. These results indicate the importance of the effect of analyte length, temperature and the environment surrounding the ligand for accomplishing an all or none single base mismatched detection system.

Original languageEnglish
Pages (from-to)23-31
Number of pages9
JournalColloids and Surfaces B: Biointerfaces
Volume27
Issue number1
DOIs
Publication statusPublished - 2003 Jan 1

Fingerprint

Peptide Nucleic Acids
Surface Plasmon Resonance
Surface plasmon resonance
Biosensing Techniques
nucleic acids
mutations
bioinstrumentation
surface plasmon resonance
Point Mutation
Biosensors
peptides
DNA
deoxyribonucleic acid
Ligands
ligands
Temperature
ras Genes
probes
sensors
Sensors

Keywords

  • DNA diagnosis
  • DNA hybridization
  • K-ras codon12
  • Peptide nucleic acid
  • Point mutation
  • Surface plasmon resonance

ASJC Scopus subject areas

  • Biotechnology
  • Colloid and Surface Chemistry
  • Physical and Theoretical Chemistry
  • Surfaces and Interfaces

Cite this

Detection of a K-ras point mutation employing peptide nucleic acid at the surface of a SPR biosensor. / Sato, Yasunobu; Fujimoto, Keiji; Kawaguchi, Haruma.

In: Colloids and Surfaces B: Biointerfaces, Vol. 27, No. 1, 01.01.2003, p. 23-31.

Research output: Contribution to journalArticle

@article{735154a147a546309bb183f4884d73c7,
title = "Detection of a K-ras point mutation employing peptide nucleic acid at the surface of a SPR biosensor",
abstract = "A fundamental study of the usage of peptide nucleic acid (PNA) and DNA at the surface of a surface plasmon resonance (SPR) biosensor for detecting a specific point mutation in the K-ras gene for early detection of cancers is reported. The extents of hybridization using DNA analytes of different length which are targets were studied at 25 and 40°C. The 15mer PNA ligand immobilized to the surface of the sensor had a high ability to detect a 11mer DNA analyte point mutation at 25 and 40°C. On the contrary, when DNA was used as a ligand, detection was only confirmed at 40°C which indicates that the difference of melting temperature (Tm) between the complementary and mismatched analyte was a key factor for point mutation detection. This also implied the decrease of Tm by immobilizing a probe to a surface since it was below the one in solution. Therefore, effects from the surface are seen in this system and the surrounding environment makes a difference to the discrimination process. To understand the process in detail, kinetic analysis was performed and association constants were found to be larger for DNA ligands implying the effect coming from the sensor surface. In this case negative charges of the carboxyl groups of the matrix had a tendency to repel negatively charged DNA ligands. Affinity constants (KA) were calculated and as expected, the ratio of KA between the complementary and mismatched analyte was of the order of 102 for PNA interacting with 11mer analytes, making it a promising probe for genetic diagnosis. These results indicate the importance of the effect of analyte length, temperature and the environment surrounding the ligand for accomplishing an all or none single base mismatched detection system.",
keywords = "DNA diagnosis, DNA hybridization, K-ras codon12, Peptide nucleic acid, Point mutation, Surface plasmon resonance",
author = "Yasunobu Sato and Keiji Fujimoto and Haruma Kawaguchi",
year = "2003",
month = "1",
day = "1",
doi = "10.1016/S0927-7765(02)00027-9",
language = "English",
volume = "27",
pages = "23--31",
journal = "Colloids and Surfaces B: Biointerfaces",
issn = "0927-7765",
publisher = "Elsevier",
number = "1",

}

TY - JOUR

T1 - Detection of a K-ras point mutation employing peptide nucleic acid at the surface of a SPR biosensor

AU - Sato, Yasunobu

AU - Fujimoto, Keiji

AU - Kawaguchi, Haruma

PY - 2003/1/1

Y1 - 2003/1/1

N2 - A fundamental study of the usage of peptide nucleic acid (PNA) and DNA at the surface of a surface plasmon resonance (SPR) biosensor for detecting a specific point mutation in the K-ras gene for early detection of cancers is reported. The extents of hybridization using DNA analytes of different length which are targets were studied at 25 and 40°C. The 15mer PNA ligand immobilized to the surface of the sensor had a high ability to detect a 11mer DNA analyte point mutation at 25 and 40°C. On the contrary, when DNA was used as a ligand, detection was only confirmed at 40°C which indicates that the difference of melting temperature (Tm) between the complementary and mismatched analyte was a key factor for point mutation detection. This also implied the decrease of Tm by immobilizing a probe to a surface since it was below the one in solution. Therefore, effects from the surface are seen in this system and the surrounding environment makes a difference to the discrimination process. To understand the process in detail, kinetic analysis was performed and association constants were found to be larger for DNA ligands implying the effect coming from the sensor surface. In this case negative charges of the carboxyl groups of the matrix had a tendency to repel negatively charged DNA ligands. Affinity constants (KA) were calculated and as expected, the ratio of KA between the complementary and mismatched analyte was of the order of 102 for PNA interacting with 11mer analytes, making it a promising probe for genetic diagnosis. These results indicate the importance of the effect of analyte length, temperature and the environment surrounding the ligand for accomplishing an all or none single base mismatched detection system.

AB - A fundamental study of the usage of peptide nucleic acid (PNA) and DNA at the surface of a surface plasmon resonance (SPR) biosensor for detecting a specific point mutation in the K-ras gene for early detection of cancers is reported. The extents of hybridization using DNA analytes of different length which are targets were studied at 25 and 40°C. The 15mer PNA ligand immobilized to the surface of the sensor had a high ability to detect a 11mer DNA analyte point mutation at 25 and 40°C. On the contrary, when DNA was used as a ligand, detection was only confirmed at 40°C which indicates that the difference of melting temperature (Tm) between the complementary and mismatched analyte was a key factor for point mutation detection. This also implied the decrease of Tm by immobilizing a probe to a surface since it was below the one in solution. Therefore, effects from the surface are seen in this system and the surrounding environment makes a difference to the discrimination process. To understand the process in detail, kinetic analysis was performed and association constants were found to be larger for DNA ligands implying the effect coming from the sensor surface. In this case negative charges of the carboxyl groups of the matrix had a tendency to repel negatively charged DNA ligands. Affinity constants (KA) were calculated and as expected, the ratio of KA between the complementary and mismatched analyte was of the order of 102 for PNA interacting with 11mer analytes, making it a promising probe for genetic diagnosis. These results indicate the importance of the effect of analyte length, temperature and the environment surrounding the ligand for accomplishing an all or none single base mismatched detection system.

KW - DNA diagnosis

KW - DNA hybridization

KW - K-ras codon12

KW - Peptide nucleic acid

KW - Point mutation

KW - Surface plasmon resonance

UR - http://www.scopus.com/inward/record.url?scp=0037210199&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037210199&partnerID=8YFLogxK

U2 - 10.1016/S0927-7765(02)00027-9

DO - 10.1016/S0927-7765(02)00027-9

M3 - Article

VL - 27

SP - 23

EP - 31

JO - Colloids and Surfaces B: Biointerfaces

JF - Colloids and Surfaces B: Biointerfaces

SN - 0927-7765

IS - 1

ER -