学位の種別: 課程博士審査委員会委員 : (主査)東京大学教授 前田 瑞夫, 東京大学教授 伊藤 耕三, 東京大学教授 佐々木 裕次, 東京大学教授 竹谷 純一, 東京大学教授 船津 高

卜, 同

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論 文 の 内 容 の 要 旨 
 
 
 
論文題目    Functionalized nanoparticles for sensing and separation of biomolecules 
          （機能性ナノ粒子の生体分子検出・分離への応用） 
 
      氏  名    卜 同 
 
1 Introduction  
Nanoparticle (NP) refers to ultrafine particle with a dimension ranging from 1 to 100 nm. The small size of 
NPs results in quantum confinement effects and consequently in the tunable optical and electronic properties by 
changing the size, shape, surface chemistry, or aggregation state. These special physical properties of NPs opens 
up numerous research directions for medicine and life sciences, includes the use of NPs in drug delivery, cell 
imaging, and the NP-assisted gene therapy, just to name a few. 
However, while some of the nanomaterial-based technologies have been applied to clinical settings; many 
nanotechnologies are still under experimental stage due to various drawbacks such as low detection sensitivity, 
lack of specificity, et al. In my study, application-oriented study of functional NP was explored in a way to reach 
a biomedical design target. Microscopic method was employed to improve the sensitivity of gold nanoparticle 
(AuNP)-based DNA mutation detection; antibody-conjugated AuNPs was designed and synthesized for the 
detection of amyloid protein aggregation. Furthermore, a ferromagnetic NP was used in the selective clearance 
of amyloid aggregates from monomers.  
  
2 Detection of DNA induced gold nanoparticle aggregation with dark field imaging  
Among various metal particles, AuNPs have attracted attention due to their advantageous properties 
involving stability, activity, conductivity, and surface chemistry. In particular, assemblies of AuNPs provide 
optical and electrical properties that are distinct from individual particles. For example, aggregation of AuNPs 
accompanied by the surface plasmon shift can be recognized by a clear color change (from red to purple), which 
enables potential applications in sensors for DNA, heavy metal ions and proteins
1
. In this study, single molecule 
approach using dark field microscopy (DFM) was applied to examine AuNPs aggregation allowing the highly 
sensitive detection possible. 
The single-stranded probe DNA was immobilized on the surface of AuNPs through Au-S bond formation 
(ssDNA-AuNPs). Various amounts of complementary ssDNA were added into the ssDNA-AuNPs dispersion 
solution in the presence of 1 M NaCl. For the DFM analysis, the AuNPs samples were deposited onto 
glass-slides. DFM images were taken using microscope equipped with dark field condenser, and the size of 
Fig. 1 Schematic diagram illustrating procedures of detection of complementary DNA induced AuNP aggregation by DFM 
 
AuNPs aggregates was estimated by the intensity of each spot in DFM images. The change in the intensity 
histograms was used to determine the limit of detection (LOD) 
of the target ssDNA by 3σ criterion method and to investigate 
AuNPs aggregation models.  
As shown in Fig. 2a, the dispersed (left) and aggregated 
AuNPs (right) were identified in DFM images as spots. Figure 
2b shows intensity histograms of dispersed and aggregated 
AuNPs induced by addition of various amounts of target 
ssDNA, which indicate the size and number of each AuNPs 
aggregation. As shown in Fig. 2c, the addition of increasing 
concentrations of target ssDNA into the ssDNA-AuNP solution 
resulted in particles with higher intensities. The LOD was 
determined as low as 100 fM. 
Taking advantage of single-particle level measurement, 
the dsDNA-AuNP aggregation model was investigated by 
studying the assembly dynamics using DFM. The two NPs 
aggregate growth models, namely, particle-cluster and 
cluster-cluster models, were proposed based on modelling of 
theoretical phenomena and computer simulations (Fig.3a)
2
. 
Although it was difficult to confirm the hypothesis 
experimentally due to the heterogeneity of NPs aggregates, this 
problem can be overcome by studying the aggregate at 
single-cluster level. First, the aggregation time scale was roughly 
estimated by DLS (Fig.3b, open triangle). The size of aggregation 
indicates that the aggregation process is still in progress after 2 
hours of incubation. The change of the ratio of monomer AuNPs 
over time after the addition of 50 nM of target ssDNA was then 
estimated with DFM as described above (Fig. 3b closed circle).  
The result indicates that monomer-cluster aggregation model is 
unlikely to account for our results, indirectly suggesting that 
cluster-cluster aggregation is the dominant model for 
dsDNA-AuNP aggregation at the early stage. 
In conclusion, we have demonstrated for the first time that 
DFM can be utilized for the highly sensitive detection of DNA 
induced AuNPs aggregation at the single-cluster level. The 
sensitivity was much better than that obtained with conventional 
methods such as color change, UV-Vis spectroscopy and DLS. 
Our DFM results also suggest that the aggregation process at the 
early stage follows the cluster-cluster aggregation model. These capabilities should open opportunities for the 
use of DFM in various analytical methods using NPs assembly.  
 
3 Dark field microscopic detection of amyloid aggregates using gold nanoparticles modified with antibody  
The amyloid beta (Aβ) protein is a 39- to 43-amino acid polypeptide that is the primary constituent of senile 
Fig. 3 (a) Schematic diagram of NPs aggregation 
models (b) DNA induced AuNPs aggregation 
dynamics.  
Fig.2 (a)DFM images of dispersed and aggregated 
AuNPs, (b)Intensity histogram of AuNPs aggregates 
observed by DFM (c)Determination of LOD  
plaques and cerebrovascular deposits in Alzheimer’s disease (AD)3. The formation of Aβ aggregates is 
considered to cause AD neuro-degeneration by affecting brain nerve cells. Therefore, detection of Aβ aggregates 
is important for early recognition of diseases. Previous study reported that amyloid aggregates can be detected by 
the precipitation of AuNPs conjugated with Aβ antibodies (Ab)4. Red-colored precipitates can be seen by 
naked-eye by addition of 5 µM Aβ aggregates into AuNP-antibody (Ab-AuNP) colloid. However, this detection 
method is not sensitive enough for practical clinical use. In an aim to achieve higher sensitivity of Aβ aggregates 
detection, DFM was utilized to detect the Aβ aggregates-induced AuNPs assembly.  
Aβ fibril was prepared as previously described with some modification5. Anti-Aβ antibody was 
immobilized onto the negatively charged AuNPs surface as reported
4
. The Ab-AuNPs were treated with bovine 
serum albumin (BSA) for dispersibility in buffer. The prepared Ab-AuNP were mixed with Aβ monomer, and 
fibril solution at certain concentrations, and incubated at room temperature for 1 h. The absorbance and size 
change of AuNPs were determined by UV-Vis spectroscopy and DLS, respectively. DFM images of AuNPs 
samples were taken as described above.   
After addition of Aβ monomer and fibril at the 
same concentration, there was no color change 
distinguishable by naked eye and there was no peak-shift 
in the result of UV-vis spectroscopy (Fig. 5a and b). 
However, as shown in Fig. 5c, mixture of Ab-AuNP with 
fibril sample showed much brighter spots in DFM 
images than Ab-AuNP sample with addition of monomer. 
The addition of increasing concentrations of target Aβ 
oligomer/fibril into the Ab-AuNP solution resulted in 
particles with higher intensities. Similar to the method 
stated above, the LOD was determined as 7 pM for 
oligomer and 39 pM for fibril detection. 
 
4 Magnetic nanoparticles for selective amyloid aggregates adsorption and separation 
As stated above, both oligomeric and fibrillar Aβ aggregates are targets for therapy design in AD. 
Interaction between nano-materials and Aβ protein has attracted increasing attention. In this chapter, we explored 
the use of NPs in distinguishing and separating selectively the aggregates from monomers.  
Fig.4 Schematic diagram illustrating the procedures of DFM detection of Abeta aggregates using AuNP-antibody  
 
Fig. 5 Aβ monomer and fibril interaction with AuNP with  
antibody immobilized on the surface. (a) Color change; (b) 
UV-Vis spectroscopy; (c) DFM images 
Ferromagnetic NPs PolyMAPTAC (poly[3-(methacryloyl amino)propyl] trimethylammonium chloride) 
Co/C (MC-NP) was employed. The selective binding and separation of aggregates using MC-NPs was confirmed 
by dot blot (monomer and fibril) and Western Blot (oligomer). 
As shown in Fig. 7a, when 12.5 μM of Aβ monomer and 
fibrils were incubated with 50 μg/mL MC-NPs for 1 h, no 
fibril was left in the supernatant, while no significant change 
of the amount of monomer in the supernatant observed. When 
MC-NPs were added into the mixture of oligomer and 
monomers, oligomer was clearly removed, and monomers 
were left without absorbing to MC-NPs (Fig. 7b). These 
results indicate that MC-NPs could selectively bind to Aβ 
fibrils and oligomers. Interaction between MC-NPs and fibrils 
and oligomers was confirmed by TEM observation (Fig. 7c). 
The study of adsorption kinetics showed that the adsorption of 
fibrils onto NPs surface is a rapid process. The adsorption 
capacity of Aβ fibril by MC-NPs was determined as 1:2 
(weight) after 1 h incubation (Fig. 8). This result implies 
potential application of MC-NPs in the diagnosis and 
treatment of various diseases related to amyloid aggregates.  
 
5 Conclusion and perspectives  
In this study, the potential of NP in biomedical applications have been explored: SNPs in DNA can be 
detected using AuNPs with high sensitivity; Aβ aggregates that related to AD can not only be early sensed using 
antibody immobilized AuNPs, but also be clarified by magnetic MC-NP in solution. Successful development in 
these studies will aid the growth of biomedical industry as well as improving the quality of life in the population. 
 
Reference:  
1. (a) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J.,. Nature 1996, 382, 607-609; (b) Sato, K.; Hosokawa, K.; 
Maeda, M., J. Am. Chem. Soc. 2003, 125 (27), 8102-8103. 
2. Hemker, D. J.; Frank, C. W., Macromolecules 1990, 23 (20), 4404-4410. 
3. Yankner, B. A.; Duffy, L. K.; Kirschner, D. A., Science 1990, 250 (4978), 279-282. 
4. Sakono, M.; Zako, T.; Maeda, M., Anal. Sci. 2011, 28 (1), 73-73. 
5. Kayed, R.; Head, E.; Sarsoza, F.; Saing, T.; Cotman, C. W.; Necula, M.; Margol, L.; Wu, J.; Breydo, L.; Thompson, J. L., 
Mol Neurodegener 2007, 2 (18), 18. 
+NP 
Fig. 6 Schematic diagram illustrating the procedures of selective Abeta aggregates adsorption on MC-NPs 
Fig. 7  Interaction between Aβ protein and MC-NPs. 
 (a) dot blot; (b) WB; (c) TEM images 
Fig. 8 Characterization of adsorption of monomer 
and fibrils on MC-NP  


Functionalized nanoparticles for sensing and separation of biomolecules

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Functionalized nanoparticles for sensing and separation of biomolecules

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