Fiber Optic Particle Plasmon Resonance Biosensor for Label-Free Detection of Nucleic Acids and Its Application to HLA-B27 mRNA Detection in Patients with Ankylosing Spondylitis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents and Materials
2.2. Preparation of AuNP-Modified Optical Fibers
2.3. Bioconjufation of ssDNA Probe on AuNP-Modified Optical Fibers
- Thiolated H5-5′ ssDNA probe (30-mer base with 20-mer adenine spacer)
- HS-(CH2)6-A20-5′-TCCCT AGCAC TGGCA ATCAT GGTAG CTGGT-3′
- Thiolated H5-3′ ssDNA probe (30-mer base with 20-mer adenine spacer)
- 5′-TCCCT AGCAC TGGCA ATCAT GGTAG CTGGT-3′-A20-(CH2)6-SH
2.4. Fabrication of Sensor Chips
2.5. Biosensing System and Measurements
2.6. Relative Quantitation of mRNA-B27 in Plasma by Real Time RT-PCR
2.7. Analysis of H5 RNA Samples
- 60-mer target H5 ssDNA (60-mer, the underlined part is complementary to the probe),3′-GTTAA ATAAG TTGTC ACCGC TCAAG GGATC GTGAC CGTTA GTACC ATCGA CCAGA TAGAA-5′
- 30-mer target H5 ssDNA (30-mer, perfect match to the probe),3′-AGGGA TCGTG ACCGT TAGTA CCATC GACCA-5′
- 138-mer target H5 ssRNA (138-mer, the underlined part is complementary to the probe),5′-GUGUU UUUAA CUACA AUCUG AACUC ACAAA UUUAA AUGCA AAUUC UGCAU UGUAA CGAUC CAUUG GAGCA CAUCC AUAAA GAUAG ACCAG CUACC AUGAU UGCCA GUGCU AGGGA ACUCG CCACU GUUGA AUAAA UUG-3′
2.8. Analysis of HLA-B27 mRNA Samples
- Thiolated HLA-B27 ssDNA probe (19-mer base with 20-mer thymine spacer, MDBio, Taipei, Taiwan)
- HS-(CH2)6-5′-T20-CCTGG GCTGG CTCCC ACTC-3′.
3. Results and Discussion
3.1. Construction of Sensor Layer
3.2. Label-Free Detection of DNA-DNA Hybridization
3.3. Label-Free Detection of DNA-RNA Hybridization Using H5 Conserved Sequence as Model
3.4. Label-Free Detection of HLA-B27 mRNA in Real Clinical Samples and Comparative Study by Real-Time RT-PCR
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Name | Sequence (5′→3′), functional at 5′ end: HS-(CH2)6-ssDNA |
---|---|
19-mer probe | CCTGG GCTGG CTCCC ACTC |
21-mer probe | AGATC AGTGC GTCTG TACTA G |
23-mer probe | AGATC AGTGC GTCTG TACTA GCA |
25-mer probe | AGATC AGTGC GTCTG TACTA GCACA |
27-mer probe | ATGGG CGGCA TGAAC CGGAG GCCCA TC |
Name | Sequence (3′→5′) |
19-mer target | GGACC CGACC GAGGG TGAG |
21-mer target | TCTAG TCACG CAGAC ATGAT C |
23-mer target | TCTAG TCACG CAGAC ATGAT CGT |
25-mer target | TCTAG TCACG CAGAC ATGAT CGTGT |
27-mer target | TACCC GCCGT ACTTG GCCTC CGGGT AG |
27-mer target (nc) 1 | CTAGC TAGCT AGCTA GCTAG CTAGC TA |
Oligonucleotide Length (bp) | Molecular Weight (Da) | Binding Constant (Ka) | LOD |
---|---|---|---|
19-mer | 5912.9 | 7.2 × 106 M−1 | 2.4 × 10−9 M |
21-mer | 6657.5 | 2.5 × 107 M−1 | 4.9 × 10−10 M |
23-mer | 7259.8 | 2.7 × 107 M−1 | 3.3 × 10−10 M |
25-mer | 7862.2 | 4.5 × 107 M−1 | 1.1 × 10−10 M |
27-mer | 8531.6 | 8.8 × 107 M−1 | 9.6 × 10−11 M |
Probe | H5-5’ ssDNA Probe | H5-3’ ssDNA Probe | ||
---|---|---|---|---|
Target | 30-mer ssDNA | 60-mer ssDNA | 60-mer ssDNA | 138-mer RNA |
LOD | 1.9 × 10−10 M | 1.4 × 10−8 M | 1.2 × 10−10 M | 1.1 × 10−10 M |
Ka | 9.8 × 108 M−1 | 1.3 × 107 M−1 | 2.9 × 108 M−1 | 6.4 × 108 M−1 |
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Tseng, Y.-T.; Li, W.-Y.; Yu, Y.-W.; Chiang, C.-Y.; Liu, S.-Q.; Chau, L.-K.; Lai, N.-S.; Chou, C.-C. Fiber Optic Particle Plasmon Resonance Biosensor for Label-Free Detection of Nucleic Acids and Its Application to HLA-B27 mRNA Detection in Patients with Ankylosing Spondylitis. Sensors 2020, 20, 3137. https://doi.org/10.3390/s20113137
Tseng Y-T, Li W-Y, Yu Y-W, Chiang C-Y, Liu S-Q, Chau L-K, Lai N-S, Chou C-C. Fiber Optic Particle Plasmon Resonance Biosensor for Label-Free Detection of Nucleic Acids and Its Application to HLA-B27 mRNA Detection in Patients with Ankylosing Spondylitis. Sensors. 2020; 20(11):3137. https://doi.org/10.3390/s20113137
Chicago/Turabian StyleTseng, Yen-Ta, Wan-Yun Li, Ya-Wen Yu, Chang-Yue Chiang, Su-Qin Liu, Lai-Kwan Chau, Ning-Sheng Lai, and Cheng-Chung Chou. 2020. "Fiber Optic Particle Plasmon Resonance Biosensor for Label-Free Detection of Nucleic Acids and Its Application to HLA-B27 mRNA Detection in Patients with Ankylosing Spondylitis" Sensors 20, no. 11: 3137. https://doi.org/10.3390/s20113137
APA StyleTseng, Y.-T., Li, W.-Y., Yu, Y.-W., Chiang, C.-Y., Liu, S.-Q., Chau, L.-K., Lai, N.-S., & Chou, C.-C. (2020). Fiber Optic Particle Plasmon Resonance Biosensor for Label-Free Detection of Nucleic Acids and Its Application to HLA-B27 mRNA Detection in Patients with Ankylosing Spondylitis. Sensors, 20(11), 3137. https://doi.org/10.3390/s20113137