Zeptomole Biosensing of DNA with Flexible Selectivity Based on

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Zeptomole Biosensing of DNA with Flexible Selectivity Based on Acoustic Levitation of a Single Microsphere Binding Gold Nanoparticles by Hybridization Akihisa Miyagawa, Makoto Harada, and Tetsuo Okada ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00748 • Publication Date (Web): 28 Aug 2018 Downloaded from http://pubs.acs.org on August 29, 2018

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Zeptomole Biosensing of DNA with Flexible Selectivity Based on Acoustic Levitation of a Single Microsphere Binding Gold Nanoparticles by Hybridization

Akihisa Miyagawa, Makoto Harada, and Tetsuo Okada* Department of Chemistry, Tokyo Institute of Technology, Meguro-ku, Tokyo 152-8551, Japan

Phone and Fax: +81-3-5734-2612 Email; [email protected]

1 ACS Paragon Plus Environment

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Abstract A novel scheme for DNA sensing at the zeptomole level is presented, based on the levitation of a single microsphere in a combined acoustic-gravitational (CAG) field. The levitation of a microsphere in the field is predominantly determined by its density. Capture and reporter probe DNAs are anchored on polymethyl methacrylate microsphere (PMMA) and gold nanoparticles (AuNPs), respectively, and a target DNA induces

the

binding

of

AuNPs

on

PMMA.

This

interparticle

sandwich

DNA-hybridization induces density increase in PMMA, which is detected as a shift in the levitation coordinate in the CAG field. The reporter DNAs are designed based on base-pair (bp) number selectivity, which is evaluated using direct interparticle hybridization between DNA-bound PMMA and complementary DNA-anchored AuNPs. Interestingly, the bp-number selectivity can be enlarged by lowering the reactant concentrations. Thus, the threshold bp, at which no density change is detected, can be adjusted by controlling the reactant concentrations. This strategy is extended to the sensing of HIV-2 DNA and single nucleotide polymorphism (SNP) detection of the KRAS gene by sandwich hybridization. In SNP detection, the present method selectively distinguishes wild-type DNA from mutant DNA differing by one nucleotide in the 21-nucleotide sequence by optimizing the lengths of probe DNAs and particle


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concentrations. This approach allows the detection of 1000 DNA molecules.

Keywords; DNA hybridization; Density of microparticle; Acoustic radiation force; Selectivity adjustment; Single nucleotide polymorphism.


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DNA is a biomolecule that plays critical roles in many processes such as storing and expressing genetic information.1-2 Sensitive detection of DNA is important in many fields such as clinical diagnosis, gene therapy, food safety, environmental analysis, forensic analysis, and drug development.2-4 The polymerase chain reaction (PCR) is often employed to increase the population of target DNA. However, requirements for precise thermal control and long reaction times restrict its practical application.5 Thus, sensitive quick methods that do not rely on PCR amplification of the target DNAs are desired. A number of detection schemes have been developed using fluorescence spectrometry, electrochemistry, colorimetry, magnetic separation, surface-plasmon resonance, and surface-enhanced Raman scattering (SERS).1,

4, 6-12

These methods

should be evaluated in terms of sensitivity, mismatch specificity, and applicability. The mismatch specificity is related to the detection of single nucleotide polymorphism (SNP). The variation in the DNA sequence that does not cause disease but is the origin of the difference in response to a particular disease is commonly referred to as polymorphism. In particular, polymorphism, in which only one nucleotide in a gene sequence is replaced by another one, is called SNP. The detection of SNP is important for disease diagnosis, prognosis, and risk assessment in drug response and toxicity.13-14


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Several flexible analytical schemes have been devised using labeling detection and are widely applied. A silica nanoparticle (NP)-based DNA assay was, for example, developed by Wang and Liu.15 A chromophore (C*)-labeled target DNA formed a duplex with complementary DNA immobilized on silica NPs. A cationic conjugated polymer (CCP) was then added to the silica NPs, which caused fluorescence resonance energy transfer from CCP to C*. The detection limit of this method was 10 pM. Although labeling detection is effective in many cases, labeling processes change the physicochemical properties of the target.16 SERS is an attractive technique for DNA sensing because it allows highly sensitive label-free detection. Kang et al. reported a SERS detection scheme using gold nanowires (AuNWs) and gold NPs (AuNPs) with detection limit 10 pM.17 In this method, the target DNA caused the assembly of probe DNA-anchored AuNW with reporter DNA-bound AuNP through complementary hybridization. A SERS signal from a Raman-probe introduced at a terminal of the reporter DNA was detected when AuNPs and AuNWs were appropriately assembled in the presence of target DNAs. The application of this approach is restricted to relatively long DNAs. The size and shape-dependent optical properties NPs have also been utilized to design DNA analyses.12, 18 Maeda et al. reported highly sensitive detection using the


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non-cross-linking aggregation of AuNPs.19 When probe-anchored AuNPs were aggregated in the presence of complementary target DNA, the solution color turned blue. However, no color change occurred when a mismatch was involved near the terminal of the DNA. This method cannot detect a mismatch at the second or farther base pair from the terminal. Jio et al. reported the electrochemical impedance detection of DNA related to a specific mutation in cauliflower mosaic virus 35S.20 The electrochemical impedance on TiO2 NPs/conductive polymer-modified carbon paste electrode decreased with increasing target DNA concentration in this case. Nagaoka et al. developed an AuNP array for label-free DNA detection.21-22 Probe DNAs were anchored on AuNPs, which formed an AuNP network on an interdigital Pt electrode. Complete hybridization between the probe and target DNAs lowered the conductivity measured between Pt electrodes. Mismatches in DNA sequence were detectable because conductivity decrease became smaller in such cases. The detection limits of these electric detections are not very low, typically ranging from nM to µM. Although some other electrochemical methods have also been proposed for SNP detection,23-27 some have limited detectability or low applicability due to the requirement of a special technique. Capillary electrophoresis is also a powerful technique for SNP detection.28-29 Takarada and his coworkers developed affinity electrophoresis using a copolymer,


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which has different affinities to wild- and mutant-type DNA, as the additive. The affinities can be controlled by the MgCl2 concentration in a running solution and temperature. Although this method is flexible and high SNP specificity, fluorescence labeling is required. Because of intrinsic poor detectability of capillary electrophoresis, sample concentration should be higher than nM. In a previous study, we presented a detection scheme based on levitation coordinate

measurements

of

a

single

microparticle

(MP)

in

a

combined

acoustic-gravitational (CAG) field.30 This field recognized the acoustic properties of particles such as density and compressibility. The particles with different acoustic properties levitated at different coordinates in this field. If a change in acoustic property is induced by reaction on the MP, the levitation coordinate changes accordingly. We utilized density change induced by the binding of AuNPs to MPs through the avidin-biotin reaction. In the present study, we extend this scheme to the label-free detection of DNA. Target DNA induces the binding of reporter DNA-bound AuNPs with a capture DNA-bound microsphere. Selectivity of this approach can be managed by the matching base-pair (bp) number and by the reactant concentration. The present strategy allows us to design versatile trace analyses with high selectivity.


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Methods and Materials The experimental setup (Figure S1) was the same as that used in our previous studies.30-34 Details are given in the Supporting Information. Calboxyl-functionalized polymethyl methacrylate microparticles (PMMAs; 9.57 ± 0.21 µm diameter, 20 µmol g-1 carboxyl groups) were purchased from Microparticle GmbH (Berlin, Germany). Calboxyl-functionalized gold nanoparticles (AuNPs; 100 nm diameter, about 255 µmol g-1 carboxyl groups) were purchased from Cytodiafnotics (Burlington, Canada). Oligonucleotides, which were purified by HPLC, were purchased from Fasmac Co., Ltd. (Kanagawa, Japan). The sequences of nucleotides used in this work are shown in Tables S1-S3. The number of nucleotides in a single-strand DNA (ssDNA) is denoted nt. Amino-terminated probe ssDNAs were covalently conjugated with carboxyl-functionalized PMMAs or carboxyl-functionalized AuNPs; the resulting particles are hereafter denoted D-PMMAs and D-AuNPs, respectively.

Results and Discussion Direct binding of D-AuNP on D-PMMA through interparticle complementary DNA hybridization The levitation coordinate of a microsphere, z, in the CAG field is given by 30-34 z=

(ρ − ρ ′)gλ  λ sin −1   4π  AEac 2π 

(1) 8 ACS Paragon Plus Environment

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A=

5ρ ′ − 2 ρ γ ′ − 2ρ ′ + ρ γ

(2)

where λ is the ultrasound wavelength; Eac the average ultrasound energy density; g the gravitational acceleration; ρ and γ are the density and compressibility of the medium (water in the present case), respectively; and the primes in the equations represent the corresponding properties of the microsphere. Eq. (1) suggests that z depends on particle density and compressibility but is independent of its size. Thus, we can evaluate the acoustic properties of the microsphere from the levitation position in the CAG field. In this study, the acoustic properties of single PMMA particles were modified by the binding of AuNP through DNA hybridization, schematically shown in Figure 1. Equation (1) indicates that the levitation position of a particle in the CAG field is lowered with increasing density or compressibility. However, our previous work indicated that z was more sensitive to change in density of a microsphere than compressibility for the binding between a polymer microsphere and AuNPs.30 Therefore, the levitation coordinate of PMMA could be discussed as a function of its density, modified by AuNP-binding. However, because of the heterogeneity of the density and compressibility of the PMMA particle binding AuNPs, this particle does not obey Equation (1), which was derived for a homogeneous particle, in a rigorous sense. The bp number specificity was studied using direct interparticle hybridization


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between a D-PMMA and D-AuNPs based on the scheme illustrated in Figure 1A. D(13)-PMMAs (nt of ssDNA in parentheses) were treated with complementary D(7-12)-AuNP. The number ratio of D-AuNPs to D-PMMAs (rAuNP/PMMA) was varied in the range 1699−8497. The levitation coordinate shift, ∆z, between untreated and AuNP-bound PMMA was measured in the CAG field. The levitation coordinate of a PMMA, which was trapped at V = 18 V, was measured at V = 5.4 V, and this procedures was repeated five times. The average ∆z value are reported together with the standard deviation in the subsequent figures. Figure 2 shows that the relationship between ∆z and rAuNP/PMMA for 7−12 base pair (bp) DNA was formed by direct interparticle hybridization. The number concentration of D-PMMA particles (nPMMA) in the reaction mixture was kept constant at 1.4 × 104 mL-1. For 8−12-bp, ∆z increased linearly with increase in rAuNP/PMMA, suggesting that the scheme illustrated in Figure 1 worked as expected. Although Equation (1) predicts ∆z/rAuNP/PMMA = 0.022 µm, the actual value (sensitivity) was smaller than the prediction; e.g. ∆z/rAuNP/PMMA = 0.0078 µm for 12-bp from Figure 2. This suggests the limited applicability of Equation (1) for the reason noted above and also possibility that all of the added AuNPs are not bound to the PMMA. Sensitivity decreased with decreasing bp because DNA hybridization became weaker. In actuality, the binding of D(7)-AuNPs with D-PMMA was so weak that


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change in z due to AuNP binding was not detected. The detection limits in this condition were 400, 800, 900, 2000, and 5200 rAuNP/PMMA for 12-, 11-, 10-, 9-, and 8-bp, respectively. The binding of AuNPs on PMMA in the equilibrium state naturally depends on particle concentration in a reaction mixture. The results obtained with nPMMA = 65 mL-1 are shown in Figure S2. The ∆z values decreased compared to the corresponding ones obtained at nPMMA = 1.4 × 104 mL-1. In this condition, D(8)-AuNPs gave only a negligible shift of ∆z. Thus, we could control the threshold bp at which ∆z became undetectable by adjusting nPMMA. Table 1 lists the slope of the ∆z-rAuNP/PMMA plot for each bp. We could discuss the bp selectivity of this approach based on the ratio of slopes. For 9–12-bp DNA, the s12/sbp ratio ranged from 1 to 1.5 at nPMMA = 1.4 × 104 mL-1, indicating that the DNA length could not be clearly resolved in this condition. Interestingly, the bp-selectivity could be enlarged by decreasing nPMMA. Low particle concentrations facilitated the dissociation of dsDNA formed through interparticle hybridization. Because this effect was more critical for dsDNA with smaller bp, selectivity was enlarged at lower nPMMA. Table 1 indicates that no binding was detected for 8-bp at nPMMA = 65 mL-1 (as stated above), and the s12/s9 and s12/s10 ratios were twice that of the corresponding values at nPMMA = 1.4 × 104 mL-1. Thus, bp-selectivity could be controlled by varying nPMMA.


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This is one of the characteristic features of the present approach. The dissociation constant of dsDNA, K, can be calculated for each bp from ∆G°, which is given by the NN model proposed by Walder and coworkers.35 For example, K for 12-bp dsDNA used in the present work was estimated to be 2.9 × 10-15. The ∆z values measured for D(7−12)AuNP-bound PMMAs at rAuNP/PMMA = 8.50 × 103 are plotted against the calculated log K in Figure 3. The results obtained with nPMMA = 1.4 × 104 and 65 mL-1 are shown in black and red symbols, respectively. Obviously, ∆z decreased with increasing K (decreasing bp). The equilibrium concentration of dsDNA for each bp could be estimated using K and concentrations. This estimation predicts the sigmoidal changes in ∆z with log K (shown by broken curves in Figure 3) where we assumed that the maximum ∆z was 65 µm and ∆z increased linearly with the number of bound AuNPs. Although the tendency was interpreted, obvious deviations were seen between the experimental values and estimations. The anchoring of DNA on a particle affected the thermodynamics of DNA hybridization. It is known that hybridization is thermodynamically enhanced when DNAs are anchored on the NP surface. A recent paper showed that this effect came from enthalpically favorable structures of DNAs on the NP surface.36 On the other hand, the rate constant of a diffusion-controlled reaction is proportional to the diffusion


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coefficients of the reactants. Because the hydrodynamic size of AuNPs used in this work was larger than those of 8−12-bp DNA molecules by two orders of magnitude, the reaction rate constant was also different by this order. Thus, the anchoring of DNA molecules on AuNPs brought about two opposing effects, which caused a shift of K in the present case from that estimated for hybridization between free solution DNAs. Solid sigmoidal curves in Figure 3 were calculated using K values that were smaller than the corresponding values estimated with the NN model by 1.5 orders of magnitude. The modified K values explain the experimental ∆z better than those estimated by the NN model. Thus, 8−12-bp dsDNA formed by interparticle hybridization was thermodynamically less stable than the corresponding one formed in free solution by ~8.5 kJ mol-1.

Label-free DNA sensing by sandwich hybridization and application to SNP detection Based on the bp number specificity revealed by direct binding studies, the label-free determination of DNAs was studied using sandwich hybridization as schematically shown in Figure 1B. The HIV-2-specific base sequence (25-nt, shown in Table S2) was chosen as target DNA. Capture probe DNA (13-nt) was anchored on PMMA and reporter probe DNA (12-nt) was anchored on AuNP to entrap the target


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DNA by interparticle hybridization. The above results of direct hybridization show that hybridization with 12−13-bp formation gave stable dsDNA in the present condition. Therefore, D(13)-PMMAs were first treated with target DNA (rDNA/PMMA = 3000– 15,000) and then with D(12)-AuNPs at constant rAuNP/PMMA of 1.55 × 104. Figure S3A shows the relationship between ∆z and rDNA/PMMA at nPMMA = 9.4 × 103 mL-1. A linear relationship was confirmed, suggesting that the scheme shown in Figure 1B worked as expected. The results demonstrated in Figures 2 and 3 indicate that interparticle hybridization became weaker as bp in the formed dsDNA decreased. The effects of probe DNA lengths were studied to assess whether the results for interparticle hybridization were directly applicable to sandwich hybridization. Two combinations of capture and reporter probes were studied: (1) 10-nt DNAs and (2) 11-nt DNAs for both capture and reporter probes. Results are summarized in Figure S3A. The slope of the ∆z-rDNA/PMMA plot became smaller as bp decreased. Dilution of the reaction mixture more effectively reduced the response from smaller bp, similar to direct hybridization. The relationship between ∆z and rDNA/PMMA at nPMMA = 65 mL-1 is given in Figure S3B. In this condition, interparticle hybridization of 20-bp dsDNA was not detected even in the presence of sufficient amount of HIV-2 target DNA. Although sensitivity reduced by


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diluting the reaction mixture, 3700 HIV-2 DNA molecules at nPMMA = 65 mL-1 were detectable. Table 2 lists the ratios of the slope for 25-bp to those for smaller bp numbers (s25/sbp).

Numerical values indicate enhanced selectivity at lower particle

concentration: s22/s25 = 0.74 and s20/s25 = 0.55 (reciprocals of the values listed in Table 2) at nPMMA = 9.4 × 103, whereas s22/s25 = 0.33 and s20/s25 = 0 at 65 mL-1. Sandwich hybridization was composed of two separate hybridizations. For example, 25-bp DNA formation involved 13-bp and 12-bp hybridizations. If the selectivity obtained for interparticle hybridization was directly applicable to that in sandwich hybridization, the latter should be represented by the multiplication of selectivity values of two component hybridizations. Using the values listed in Table 1, s22/s25 was calculated to be (s11/s12)×(s11/s12) =0.74 at nPMMA = 9.4 × 103 and 0.37 at nPMMA = 65 mL-1. Similarly, s20/s25 was calculated to be (s10/s12)×(s10/s12) = 0.55 at nPMMA = 9.4 × 103 and 0.02 at nPMMA = 65 mL-1. Thus, the selectivity obtained for direct interparticle hybridization was applicable to the design of sandwich hybridization for DNA sensing. As discussed above, the binding between a target DNA and probe DNA could be controlled by the number of the matching sequence and nPMMA. A SNP detection scheme was designed using the present approach. Part of the KRAS gene sequence, which is


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SNP related to the response of an anticancer drug, was studied as the target. The KRAS gene encodes a GTP-binding protein related to cell proliferation and tumor progression.37 Inhibition of epidermal growth factor receptor (EGFR) with monoclonal antibodies is utilized for cancer patients with a wild type KRAS gene. However, the EGFR inhibitor does not act on patients with KRAS mutation.38 Thus, identification of the genotype in the KRAS gene is important for effectively promoting cancer treatment. Figure 4 shows the principle of the detection of SNP. Both wild and mutant DNAs have identical affinities to the SNP capture probe DNA because hybridization allows the formation of complementary 12-bp dsDNA. The binding of reporter DNA with 9 nt was used for wild DNA detection; this led to hybridization that occurred for the mutant to from 8-bp dsDNA. As discussed above, the present method resolves the differences in stability between 8- and 9-bp dsDNAs. The selectivity between bp numbers was enhanced by adjusting the appropriate nPMMA. Therefore, the detection selectivity of wild DNA against mutant DNA could be maximized by managing nPMMA. Figure 5 shows ∆z- rDNA/PMMA plots at nPMMA = 9.4 × 103 mL-1 and 1.17 × 102 mL-1. Table 3 summarizes the slopes in ∆z- rDNA/PMMA plots for wild and mutant DNAs in the range nPMMA = 65–(9.4 × 103) mL-1. Selectivity toward wild DNA against mutant DNA obviously increased with decrease in nPMMA. The mutant DNA gave almost zero slope at


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nPMMA < 2.3 × 102 mL-1 whereas that of wild DNA was detectable. About 2700 wild DNA molecules were detectable at nPMMA = 2.3 × 102 mL-1 without interference from the mutant. Thus, SNP was successfully detected by controlling nPMMA and appropriately designing the reporter DNA.

Conclusion We proposed a label-free zmol detection scheme of DNA using the CAG field. Controlling selectivity toward the bp number is one of the key features of this method. Selectivity could be managed by changing particle concentrations in a reaction mixture. The critical bp-number was determined to be 8; this length was detectable in high reactant concentrations but undetectable in dilute conditions. Thus, we can control the bp-number selectivity by changing reactant concentrations. SNP detection can be designed based on this characteristic feature of the present scheme. Another advantage of this method is its high sensitivity, which originates from the detection principle in the present approach. In the present study, DNA hybridization caused AuNP binding on PMMA and induced density increase of PMMA. We could count the number of AuNPs bound to a single PMMA based on the levitation coordinate in the CAG field at AuNP number of 1000 or lower.


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There remain methodological drawbacks in the present method: particular care should be paid to particle reactions, and entrapment of a single microsphere requires techniques and skills. If the present method is integrated with a microchip in which reactions and particle entrapment are conducted, practical applications should become easier. Such studies should be carried out to make the present method more useful in practical situations.

Acknowledgment We would like to thank Drs. M. Maeda and T. Takarada (RIKEN, Japan) for helpful discussions. This work was supported by a Grant-in-Aid for Challenging Exploratory Research from the Japan Society for the Promotion of Science and the Sasakawa Scientific Research Grant from the Japan Science Society.

The Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental details; Sequences of target and probe DNAs; Experimental setup; Relationship between ∆z and rAuNP/

PMMA

in direct interparticle hybridization;

Relationships between ∆z and rDNA/PMMA in sandwich hybridization.


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The authors declare no competing financial interest.


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Conrad, S.; Wolburg, H.; Northoff, H.; Wiskirchen, J.; Weissert, R., The Use of Clinically Approved Small Particles of Iron Oxide (SPIO) for Labeling of Mesenchymal Stem Cells Aggravates Clinical Symptoms in Experimental Autoimmune Encephalomyelitis and Influences Their In Vivo Distribution. Cell Transplant. 2008, 17, 923-941. 17. Kang, T.; Yoo, S. M.; Yoon, I.; Lee, S. Y.; Kim, B., Patterned multiplex pathogen DNA detection by Au particle-on-wire SERS sensor. Nano.Lett. 2010, 10, 1189-1193. 18. Merkoci, A., Nanoparticles-based strategies for DNA, protein and cell sensors. Biosens. Bioelectron. 2010, 26, 1164-1177. 19. Sato, K.; Hosokawa, K.; Maeda, M., Rapid Aggregation of Gold Nanoparticles Induced by Non-Cross-Linking DNA Hybridization. J.Am.Chem.Soc. 2003, 125, 8102-8103. 20. Hu, Y. W.; Yang, T.; Wang, X. X.; Jiao, K., Highly sensitive indicator-free impedance sensing of DNA hybridization based on poly(m-aminobenzenesulfonic acid)/TiO2 nanosheet membranes with pulse potentiostatic method preparation. Chemistry, 2010, 16, 1992-1999. 21. Shiigi, H.; Tokonami, S.; Yakabe, H.; Nagaoka, T., Label-Free Electronic Detection


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of DNA-Hybridization on Nanogapped Gold. J.Am.Chem.Soc. 2005, 127 , 3280-3281. 22. Tokonami, S.; Shiigi, H.; Nagaoka, T., Open Bridge-Structured Gold Nanoparticle Array. Anal. Chem.2008, 80, 8071-8075. 23. Akagi, Y.; Makimura, M.; Yokoyama, Y.; Fukazawa, M.; Fujiki, S.; Kadosaki, M.; Tanino, K., Development of a ligation-based impedimetric DNA sensor for single-nucleotide

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Table 1 Slopes (s) of the ∆z-rAuNP/PMMA plots and ratios of slopes for D(9-12)-AuNPs at nPMMA.

nPMMA / mL

1.4 × 10

65

/ 10-3

µm

µm

12

7.8

0.57

4

11 10 9 8

6.6 5.7 5.2 1.2

12

σ

s12 / sbp

/ 10−3

0.50 0.57 0.55 0.85

1.2 1.4 1.5 6.5

0.85 0.73 0.67 0.15

4.6

0.58

-

-

11

2.8

0.29

1.6

0.61

10

1.9

0.22

2.4

0.41

9

1.3

0.47

3.5

0.28

8

0.1

0.35

46

0.02

bp

-1

σa

Slope (s) / 10-3

a, standard deviation (n = 5).


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Table 2

Slopes of ∆z-rAuNP/PMMA plots obtained for HIV2 DNA (20–25-bp) at different

nPMMA. nPMMA

Slope

σa

/ 10-3 µm

/ 10-3 µm

25

3.8

0.35

-

-

22

2.8

0.16

1.4

0.11

20

2.1

0.28

1.8

0.16

25

1.8

0.15

-

-

22

0.6

0.13

3.0

0.23

20

0.0

0.23

-

-

bp

/ mL-1 9.4 × 10

3

65

s25/ sbp

σa / 10-3 µm

a, Standard deviation (n = 5).

Table 3

Slopes of ∆z-rAuNP/PMMA plots obtained for wild and mutant at different nPMMA.

DNA type

wild

Slope

σa

/ 10-3 µm

/ 10-3 µm

9.4 × 103

1.7

0.37

2.3 × 102

1.5

0.37

2

1.3

0.15

0.9

0.25

9.4 × 103

0.7

0.31

2.3 × 10

2

0.2

0.33

1.2 × 10

2

-0.1

0.22

0.0

0.29

-1

nPMMA / mL

1.2 × 10 65

mutant

65 a, standard deviation (n = 5).


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Figure 1 Schematic representation of (A) the direct binding between D-PMMA and D-AuNPs by complementary DNA hybridization and (B) sandwich hybridization for label-free detection. In both cases, AuNP-binding lowers the levitation coordinate of PMMA by ∆z.


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Page 29 of 33

70

12 bp

60

11 bp

50

∆z / µm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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40

9 bp

30

10 bp 20

8 bp 10 0

7 bp -10 0

2000

4000

6000

8000

10000

rAuNP/PMMA Figure 2 Relationship between ∆z and rAuNP/ PMMA in direct interparticle hybridization for 7–12-bp dsDNA formations at nPMMA = 1.4 × 104 mL-1. Error bars represent standard deviations based on five measurements for each point.


ACS Sensors

80

12-bp 9-bp 10-bp

60

∆z / µm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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11-bp

40

20

8-bp 7-bp

0

-8

-10

-12

-14

-16

log K Figure 3 Relationships between ∆z at rAuNP/ PMMA = 8697 and log K (K: calculated duplex dissociation constant). PMMA concentration, nPMMA = 1.4 × 104 (black) and 65 mL-1 (red). Broken curves were calculated using K calculated by the NN model (values taken as the abscissa) , and solid curves were calculated assuming that the actual dissociation constant was larger by 1.5 orders of magnitude than the values estimated by the NN model. For this estimation, the following assumptions were made: ∆z for the fully AuNP-loaded PMMA was 65 µm; ∆z linearly decreased with decreasing number of AuNPs bound to PMMA.


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Figure 4 Schematic representation of the principle in SNP detection.


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Figure 5 Relations between ∆z and rDNA/PMMA in SNP detection at (A) nPMMA = 9.4 × 103 mL-1 and (B) 1.17 × 102 mL-1. rAuNP/PMMA = 1.55 × 104. Error bars represent standard deviations based on five measurements for each point.


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TOC


Zeptomole Biosensing of DNA with Flexible Selectivity Based on

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