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Single Nucleotide Polymorphism Highlighted via Heterogeneous Light-Induced Dissipative Structure
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  • Shuichi Toyouchi
    Shuichi Toyouchi
    Research Institute for Light-induced Acceleration System (RILACS), Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
    Department of Physics, Graduate School of Science, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
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    Orcidhttps://orcid.org/0000-0002-4771-8015
  • Seiya Oomachi
    Seiya Oomachi
    Research Institute for Light-induced Acceleration System (RILACS), Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
    Department of Physics, Graduate School of Science, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
    Department of Materials Science, Graduate School of Engineering, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
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  • Ryoma Hasegawa
    Ryoma Hasegawa
    Research Institute for Light-induced Acceleration System (RILACS), Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
    Department of Physics, Graduate School of Science, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
    Department of Materials Science, Graduate School of Engineering, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
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  • Kota Hayashi
    Kota Hayashi
    Research Institute for Light-induced Acceleration System (RILACS), Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
    Department of Physics, Graduate School of Science, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
    Department of Materials Science, Graduate School of Engineering, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
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  • Yumiko Takagi
    Yumiko Takagi
    Research Institute for Light-induced Acceleration System (RILACS), Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
    Department of Physics, Graduate School of Science, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
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  • Mamoru Tamura
    Mamoru Tamura
    Research Institute for Light-induced Acceleration System (RILACS), Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
    Department of Materials Engineering Science, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan
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  • Shiho Tokonami*
    Shiho Tokonami
    Research Institute for Light-induced Acceleration System (RILACS), Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
    Department of Materials Science, Graduate School of Engineering, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
    *Email: [email protected]
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  • Takuya Iida*
    Takuya Iida
    Research Institute for Light-induced Acceleration System (RILACS), Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
    Department of Physics, Graduate School of Science, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0003-1313-7025
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ACS Sensors

Cite this: ACS Sens. 2025, 10, 2, 751–760
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https://doi.org/10.1021/acssensors.4c02119
Published January 23, 2025

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Abstract

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The unique characteristics of biological structures depend on the behavior of DNA sequences confined in a microscale cell under environmental fluctuations and dissipation. Here, we report a prominent difference in fluorescence from dye-modified single-stranded DNA in a light-induced assembly of DNA-functionalized heterogeneous probe particles in a microwell of several microliters in volume. Strong optical forces from the Mie scattering of microparticles accelerated hybridization, and the photothermal effect from the localized surface plasmons in gold nanoparticles enhanced specificity to reduce the fluorescence intensity of dye-modified DNA to a few %, even in a one-base mismatched sequence, enabling us to clearly highlight the single nucleotide polymorphisms in DNA. Fluorescence intensity was positively correlated with complementary DNA concentrations ranging in several tens fg/μL after only 5 min of laser irradiation. Remarkably, a total amount of DNA in an optically assembled structure of heterogeneous probe particles was estimated between 2.36 ymol (2.36 × 10–24 mol) and 2.36 amol (2.36 × 10–18 mol) in the observed concentration range. These findings can promote an innovative production method of nanocomposite structures via biological molecules and biological sensing with simple strategies avoiding genetic amplification in a PCR-free manner.

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Thermal fluctuations and energy dissipation are inextricably linked in the formation of dissipative structures under nonequilibrium conditions and are especially important in the formation of ordered biological structures. (1) Since the inception of life on earth 3.5 billion years ago, optimized photoreceptors, photosynthetic antennae, and ordered structures with diverse spatial patterns have evolved under various external fields such as thermal fluctuations and ambient light. (2−4) Over the course of evolution, DNA formed a narrow space enclosed by a lipid bilayer in which information was transcribed by RNA, a system for protein production was established, and self-replication was achieved in accordance with the central dogma. (5,6) The double-helix structure of DNA elucidated in 1953, (7) and the base sequence of human DNA reported by the Human Genome Project (8) represent notable milestones in the life sciences. However, the mechanisms underlying the precise transmission of genetic information and mutations in various clinically important molecules, such as KRAS and EGFR in cancer development, remain unclear. (9,10) Recent theories indicate that quantum tunneling plays important roles in gene mutations, and the quantum nature of hydrogen bonding influences the formation of ordered structures in vivo. (11,12)
Evaluation of environmental DNA (13) is important to understand ecosystem transitions and environmental changes under climate change. Cell-free DNA (cfDNA) (14,15) and circulating tumor DNA (ctDNA) (16) have also attracted attention as important biomarkers in liquid biopsies used in the diagnosis of genetic diseases, such as cancer. In those emerging DNA sensing applications, single-nucleotide polymorphisms (SNPs) are required to be exclusively detected from wild genes or other species. Thus, simple, high-throughput genetic, and highly gene-specific measurement techniques are essential for both environmental conservation and clinical care. Many techniques have been developed to analyze the differences and mutations in DNA sequences, including the Sanger sequencing using fluorescence and electrophoresis (17) and polymerase chain reaction (PCR) based on primer-based amplification. (18) Furthermore, next-generation sequencing, which incorporates the advantages of Sanger sequencing and PCR, has enabled high-throughput analysis (19) of genetic changes in cancer, dementia, and infectious diseases, making their early diagnosis more accessible. Since the discovery of PCR in the early 1980s, PCR-based methodologies have revolutionized gene diagnostics and are currently considered the gold standard for gene analysis. However, PCR-based gene analysis is expensive, time-consuming, and requires complex instrumentation, qualified personnel, and specialized laboratories. Recent progress in next-generation sequencing has enabled the simultaneous determination of sequences of thousands to millions of DNA molecules; however, the required equipment is large and expensive, and the entire process takes hours to days. Therefore, more accessible and user-friendly technologies are needed to overcome the limitations of PCR-based methods and advance the current paradigm of diagnostics. PCR-free strategies represent promising solutions for gene analysis without the amplification step, which can substantially reduce costs and improve sequencing turnover.
In genetic sequencing, target biological materials should ideally be transported toward the observation region of a sensor in a simple and effective manner. Based on the optical tweezers proposed by Ashkin et al., (20) tiny biological and nonliving materials can be precisely manipulated through electromagnetic interactions driven by light-induced force (LIF). This technology can nondestructively capture and extend a long DNA strand by trapping and moving a microparticle linked to the DNA. (21) Optical manipulation technology has been widely applied in nanoscale physics, chemistry, and biology. (22−24) However, the working range of optical tweezers is limited to the laser-irradiated area. In contrast, by optically trapping metal nanostructures with localized surface plasmons, light-induced convection (LIC) is generated over the liquid sample through photothermal effects, thereby guiding and condensing dispersoids toward the observation region (25) where the synergistic effects of the LIF and LIC facilitate the optical condensation and reaction of small amounts of biological nanomaterials. (26) With specific surface-structure substrate designs, micron-scale biological objects such as bacteria can be optically condensed with a high survival rate, even by LIC arising from the photothermal effect. (27,28) Furthermore, optical condensation through light-induced acceleration of selective antigen–antibody reactions and cellular uptake can be controlled by balancing the LIF and LIC. (29−32) Paying attention to previous studies on material engineering with biological molecules, there are many reports on the construction of various assembled structures consisting of nanoparticles (NPs) via DNA hybridization, (33−35) and on the electric and nanophotonic biosensors using molecular recognition. (36,37) For example, DNA-modified gold NPs have been used for the electric detection of target DNA at 100 pmol/μL. (38) The hybridization of DNA-modified NPs and floating single-stranded DNA is accelerated by LIF mediated by LIC at the air–liquid interface. (39) Nonresonant laser irradiation is associated with little light-induced polarization and weak LIFs on NPs smaller than the light wavelength, (40) thus requiring the assistance of LIC with evaporation. The application of optical condensation is further limited by the large size variation in the assembled composites and requires local condensation at the solid–liquid interface for the stable construction of DNA-modified composite NPs.
Here, based on the light-induced acceleration system for molecular recognition, we aimed to develop a new macroscopic light-induced assembly (LIA) method for DNA-modified composites based on two types of probe particles (Figure 1A, B). In this process, DNA molecules are modified on microparticles (probe 1), on which a strong LIF acts through Mie scattering, and gold NPs (probe 2), which enhance the electric field and the photothermal effect of localized surface plasmons. We attempted to enhance LIC and LIF via the assembly of low-density NPs and target substances by capitalizing on the collective micron-scale phenomena of localized surface plasmons. In a static fluid in a microwell, probe particles were assembled at the solid–liquid interface by laser irradiation, and the target DNA was condensed by hybridization among the heterogeneous probe particles in a sequence-specific manner. A fluorescent dye modification was used to observe the target DNA trapped in the LIA via fluorescence imaging. Finally, we evaluated the suitability of our approach for quantitatively detecting target DNA at low concentrations.

Figure 1

Figure 1. Light-induced acceleration of DNA hybridization with optically assembled DNA-functionalized gold nanoparticles (DNA-AuNP, Probe 1) and polystyrene microparticles (DNA-PSMP, Probe 2) as heterogeneous probe particles for PCR-free DNA detection. (A) Schematic illustration of experimental design. An infrared laser was loosely focused near the glass–liquid interface of a homemade microwell, where probe particles and fluorescent dye-labeled target DNA molecules were transported by light-induced force (LIF) and light-induced convection (LIC) flow and then assembled. Inside the light-induced assembly (LIA), the complementary target DNA molecules bridge the heterogeneous probes by forming double-stranded DNA. AuNPs facilitate sequence-specific DNA detection by enhancing the fluorescence signal of target DNA through surface plasmon resonance and increasing the temperature inside the LIA through excellent photothermal effects. (B) Transmission images recorded ‘a’ before laser irradiation, during (b; 0 s, c; 1 min, d; 2 min, e; 3 min, f, 4 min, g; 5 min), and after the optical condensation of heterogeneous probe particles and target DNA. Images b-g were recorded by intentionally shifting the focal plane above 30 μm from the coverglass–liquid interface to expand the laser spot about 30 μm. In h, the focal plane moved to the ceiling of the microwell after laser irradiation. Scale bar: 100 μm. See also Movie S1.

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Results and Discussion

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Light-Induced Acceleration of DNA Hybridization

A mixture of DNA-modified gold nanoparticle (AuNP) Probe <I> and DNA-modified polystyrene microparticle (PSMP) Probe <IV> dispersion liquid (heterogeneous probe-particles) and target-DNA solution (7.37 pg/μL) was laser-irradiated in a homemade microwell for 5 min. For details on the probe preparations and the experiment, see the Methods. In this study, we prepared four DNA-modified probes; the DNA-modified AuNP probe <I> and <II> and the DNA-modified PSMP probe <II> and <IV>. The probe DNA sequences and the probe combinations are described in Table S1. The target DNA sequences are described in Table S2. Optical transmission images and the movie captured during laser irradiation are demonstrated in Figure 1B and Movie S1, respectively. For the transmission imaging during laser irradiation, the focal plane was intentionally shifted 30 μm above to expand the laser spot about 30 μm. We observed PSMP movement owing to LIFs (mainly the scattering force) and the gradual assembly of particles in the laser-irradiated area. The strength of LIFs exerted on the PSMPs and pushing them toward the laser-irradiation area is related to several experimental conditions, i.e. laser power density, laser wavelength, particle material (refractive index and extinction cross-section), and particle size. We estimated the strength was 36.4 pN at the ceiling coverglass of the homemade microwell (the estimation method described in the previous literatures (29,40)). Notably, probe particles were assembled in this area, and several particles were ejected to the outside of the laser-irradiation cone. This indicated that the LIFs exerted on the microparticles and the motion of the microparticles toward the laser irradiation area resulted in convection in the static fluid. Such a wide range LIC was theoretically and experimentally studied in the photothermal optical condensation of AuNPs and bacteria (26,28) and in the nonthermal optical trapping of PSMPs. (41) Because the AuNPs (d = 30 nm) and target DNA molecules were quite small, the exerted LIFs were almost negligible. However, it is assumed that the LIC may facilitate the transport of AuNPs and target DNA molecules toward the laser-irradiated area, potentially promoting their condensation within the assembly.
Figure 2A shows the typical transmission images recorded before and after 5 min laser irradiation and typical fluorescence images recorded after laser irradiation with heterogeneous probe particles. The upper, middle, and lower sections show the results with matched DNA (complementary to the probe DNA described in Table S2), mismatched DNA (perfectly mismatched to probe DNA described in Table S2), or phosphate buffer solution as a negative control (NC, without any target DNA), respectively. Regardless of the presence or absence of DNA and sequence matches or mismatches, LIA was observed, with sizes almost comparable to those of the laser-irradiated area. A remarkable difference was observed in the fluorescence measurements. For matched DNA, the fluorescence intensity (defined as the difference between the averaged brightness in the LIA and background areas) was ∼55.5. In contrast, the fluorescence intensity was ∼20.6 for the mismatched DNA and ∼18.8 for the NC, which was comparable to that of the mismatched DNA but significantly weaker than that of the matched DNA. These results indicated that the target DNA molecules were selectively trapped and condensed inside the LIA through DNA hybridization. Note that a spontaneous probe assembly and DNA localization were not observed at the laser-irradiation area in the homemade microwell without laser irradiation, even after several hours. Although a spontaneous probe assembly was observed for an unsealed mixture droplet on a coverglass, it took more than 20 min even for 7.37 ng/μL of target DNA (see Figure S1). Therefore, compared with the spontaneous probe assembly, we concluded that merely 5 min of laser irradiation condensed the DNA molecules in the LIA, and accelerated DNA hybridization by the optical condensation.

Figure 2

Figure 2. Light-induced acceleration of DNA hybridization. (A) The LIA of target DNA and heterogeneous probe particle (top) was monitored by transmission imaging using a CMOS camera before (0 s, left) and after 1064 nm continuous wave laser irradiation for 300 s (middle). Fluorescence images were captured after laser irradiation (right). The target DNA was complementary to the probe DNA sequence (Matched, 7.37 pg/μL, upper row) or fully mismatched DNA (Mismatched, 7.37 pg/μL, middle row). A negative control (target DNA 0 pg/μL) was examined under the same experimental conditions. (B) The same light-induced acceleration experiment (as in A) performed with the homogeneous probes. Fluorescence intensity (Fluo. Int.) at the LIA and background (BG) as well as the difference (Diff) between them are listed on the right side.

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We examined the optical condensation of homogeneous probe particles, comprising a mixture of two DNA-modified PSMP Probe <III> and <IV> dispersion liquids, for comparison with the heterogeneous probe particles. Figure 2B shows the typical transmission and fluorescence images of the homogeneous probe-particles. Similar to the heterogeneous probe particles, we observed LIA in the laser irradiation area regardless of match DNA, mismatch DNA, or NC. A remarkable difference in fluorescence intensity was observed between the matched DNA (∼92.1), mismatched DNA (∼21.8), and NC (∼21.4) (Figure 2B). The fluorescence intensities were comparable between mismatched DNA (Figure 2B middle) and NC (Figure 2B lower) but significantly weaker than those for matched DNA, consistent with the results for heterogeneous probe particle optical condensation, in which the target DNA molecules were selectively trapped and condensed by laser irradiation. The fluorescence intensity for matched DNA with the homogeneous probe particles was 66% higher than that with the heterogeneous probe particles. This difference is discussed in the next section. Additionally, we examined optical condensation using another homogeneous probe particle comprising a mixture of two DNA-modified AuNP probes (<I> and <II>). In this case, no LIA and condensation of the target DNA molecules were observed at the laser-irradiation area. These results indicated that the LIFs exerted on the AuNPs and target DNA molecules are weak and negligible so the LIFs can not transport the AuNPs and target DNA molecules toward the laser-irradiation area. Since it is experimentally confirmed that the AuNP probe and target DNA molecules are involved in the LIA observed in the heterogeneous probe-particle optical condensation by conducting a supporting experiment with DNA-modified AuNPs and nonmodified PSMPs instead of DNA-modified PSMP (see Figure S2), it is considered that the LIFs exerted on the PSMPs and resulting the nonthermal LIC play an important role in transporting AuNPs and target DNA molecules and the optical condensation of DNA.

DNA-Sequence Dependence Highlighted with Photothermal Effect

We examined the specificity of optical condensation with hetero- and homogeneous probe particles for the identification of sequence mismatches by detecting target DNA molecules along with complementary (full matched, FM), 1-, 2-, 4-, 6-, 8-, 10-, 12- and 24-base-mismatched (perfect mismatched) DNA (Table S2). We employed two sequences for 1-base and 2-base-mismatched DNA molecules with different mismatched base pairs or sites. Figure 3A shows the relative fluorescence intensities (normalized that obtained with the matched DNA to be 100%) plotted as a function of the number of mismatched bases. In the optical condensation of the heterogeneous probe particles, introduction of one mismatched base reduced the fluorescence intensity by 70–90%, while the second mismatched base further reduced the fluorescence intensity to 0% (comparable to the NC level, Figure 3A). Interestingly, the reduction in fluorescence intensity depended on which base pair was replaced with a mismatched base. In 1-base mismatch 1 (MM1a), in which 2G was replaced with C, the fluorescence intensity was reduced by 90% (opened red circle in Figure 3A for one mismatched base). In 1-base mismatch 2 (MM1b), in which 17A was replaced with T, the fluorescence intensity was reduced by 70% (closed red circle in Figure 3A for one mismatched base). The difference in the reduction in fluorescence intensity can be explained by the number of hydrogen bonds in the base pairs. Three hydrogen bonds were formed in the GC base pair, and two in the AT base pair. Thus, the change in enthalpy was greater in the formation of the GC base pair than that for the AT base pair, and the mismatch in the GC base pair had a larger impact on the total binding energy. In contrast, the fluorescence intensity was reduced only by ∼40% in the optical condensation of homogeneous probe particles with one mismatched base, and no mismatched base pair dependence was observed (opened and closed blue triangles in Figure 3A for one mismatched base). Six mismatched bases were required to reduce the fluorescence intensity to the NC level. These results demonstrated that optical condensation with heterogeneous probe-particles has a higher specificity in the base sequence.

Figure 3

Figure 3. Dependence of optical acceleration on DNA sequence. (A) Relative fluorescence intensity (normalized to that obtained with Matched DNA) with heterogeneous probe particles (red circles) and homogeneous probe particles (blue triangles) with varying numbers of mismatching base pairs. The error bar represents the SD (n = 3). For 1-base and 2-base-mismatched DNA, two sequences of each condition (MM1a/MM1b, and MM2a/MM2b) were evaluated. MM1a and MM2a are represented as the opened marker, and MM1b and MM2b are represented as the closed marker. (B) Schematic illustration changes in DNA structure with melting temperature (Tm). The Tm for Matched DNA is generally higher than that for Mismatched DNA (Tm’). In optical condensation using heterogeneous probe particles, the temperature inside the LIA (TLIA) is expected to increase above room temperature owing to the photothermal effect of the AuNPs. See also the DNA sequences in Table S2.

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We evaluated the differences in the sensitivity and specificity of hetero- and homogeneous probe-particle optical condensation based on the DNA melting point and photothermal conversion under laser irradiation (Figure 3B). Because DNA hybridization proceeds through hydrogen bonding, binding is weak under high temperatures. DNA molecules tend to exist in double- and single-stranded forms at temperatures lower and higher than the melting temperature (Tm). In a heated environment, DNA hybridization is subject to sequence-specific selection, depending on the binding energy and Tm. Complementary DNA sequences generally have a high Tm and maintain their double-stranded form even at high temperatures. In contrast, mismatched DNA sequences, which generally have a lower Tm’ than Tm, change to single-strand forms. Therefore, complementary DNA sequences selectively form duplex DNA at a temperature higher than Tm’ but lower than Tm. A heating strategy for a higher base sequence specificity has been employed in the PCR annealing process. Because AuNPs are good plasmonic heat sources, (42−44) the temperature inside the LIA (TLIA) is expected to increase above room temperature under laser irradiation with heterogeneous probe particles, thereby facilitating the selective progression of DNA hybridization by complementary DNA. In addition, it is assumed that the inhomogeneous temperature distribution induced by the laser irradiation and the plasmonic heating with AuNPs would result in faster diffusion and thermophoretic motion toward the outside of the LIA. This thermal effect on mobility is expected to be more prominent on smaller particles and molecules. Thus, there would be two competitive factors; on one hand, AuNPs and target DNA molecules are ejected from the LIA by thermal mobility, and on the other hand, AuNPs and target DNA molecules are captured in the LIA by DNA hybridization. For the matched DNA case, the DNA hybridization may overcome thermal mobility, while for the mismatched DNA case, thermal mobility exclusively ejects unbound AuNPs and target DNA molecules. For these reasons, higher sequence specificity was observed with heterogeneous probe particles than with homogeneous probe particles, in which the AuNPs act as a medium for photothermal conversion. This is consistent with the occasional bubble formation following optical condensation performed with heterogeneous probe particles and a higher laser power (∼800 mW; a typical transmission image is shown in Figure S3). High temperatures may reduce the sensitivity of DNA detection because double-stranded DNA may melt to single-stranded DNA at ∼ TLIA. We also theoretically confirmed that light-induced heat proportional to the absorption cross-section of each AuNP (45) can be greatly enhanced at 1064 nm as the wavelength of irradiated laser because of the spectral redshift via attractive dipole-interaction of LSPs and the broadening via plasmonic super-radiance arising from the quantum effect of interacting LSPs (46,47) when AuNPs were densely assembled (Figure S4). In contrast, homogeneous probe particles without AuNPs merely increased the temperatures inside the LIA above room temperature, and allowed less thermal mobility. Thus, most of the target DNA molecules even with a mismatched sequence remained as double-stranded DNA or remained in the LIA under laser irradiation, resulting in less specificity but higher sensitivity with homogeneous probe particles.
The high gene-specific DNA detection with the heterogeneous probe particles potentially enables us to distinguish SNPs in DNA by appropriately designing probe DNA sequences. SNPs are key genetic mutations that frequently seen in many diseases like cancer. For example, the KRAS gene, which is related to cell proliferation, has frequently observed SNPs at Codon 12, such as G12C, G12D, and G12 V. (48) Selectively detecting those SNPs from wild genes in cfDNA containing ctDNA may lead to early cancer diagnosis and prevention of recurrence. There are also recent reports that not only the detection of ctDNA from blood (serum and plasma) but also the analysis of ctDNA from nonblood sources (urine, cerebrospinal fluid, pleural or peritoneal fluid, saliva etc.) would provide a more sensitive information for particular tumor types or locations in patient body. (16) Additionally, in the emerging field of environmental DNA detection, SNPs are being investigated for species identification under climate change, pollution, and ecosystem changes. The DNA detection using optical condensation with the heterogeneous probe particles experimentally demonstrated in this work exhibits a potential for opening new rapid and highly sensitive and gene-specific detection SNPs, offering early cancer diagnosis, recurrence prevention, and environmental surveys.

Fluorescence Intensity Depends on DNA Concentration

Finally, we demonstrated that the optical condensation technique can be applied for highly sensitive and quantitative DNA detection without any amplification process (PCR-free). Figure 4A shows the fluorescence intensities obtained using the heterogeneous probe-particles as a function of the target DNA concentration for matched DNA (red circles) and mismatched DNA (blue diamonds). We observed a strong positive correlation between fluorescence intensity and target-DNA concentrations for matched DNA, while fluorescence intensities for mismatched DNA remained at the NC level, even at concentrations of several thousand fg/μL, guaranteeing high specificity. This indicated that the optical condensation technique can effectively detect the concentrations for matched target DNA in a sequence-specific manner.

Figure 4

Figure 4. Calibration curve of DNA optical condensation with heterogeneous probe particles. (A) Fluorescence intensities obtained with Matched DNA (red circles) and Mismatched DNA (blue diamonds) were plotted as a function of DNA concentration. The error bars represent the SD (n = 3). (B) Closed plot of fluorescence intensity as a function of DNA concentrations ranging from 0 to 80 fg/μL. The dashed line represents the averaged fluorescence intensity +3S.D at the NC (DNA concentration 0 fg/μL).

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The obtained fluorescence intensity (i.e., sensitivity of the DNA detection) with the heterogeneous probe-particle optical condensation may be influenced by probe storage conditions, and/or DNA sample conditions, that is temperature, pH, and contamination of other biomolecules. However, it was found that the probes and the techniques were less susceptible to probe storage temperature, and pH level and adding 10 mg/mL of bovine serum albumin (BSA) in the target DNA solution (see Figure S5). The preliminary results promise our technique is applicable for cfDNA/ctDNA detection in a clinical sample or detecting environmental DNA. The exposure time for fluorescence imaging and the photostability of the fluorescent dye (Alexa Fluor 488) may also influence the sensitivity of the detection. We examined the photostability of the dye by measuring 5 consecutive fluorescence images after an optical condensation. As a result, the dye photobleached about 12% within 1 min of light illumination for the fluorescence imaging (see Figure S6). However, the reliability of the detection is ensured by using a constant exposure time and illumination intensity.
Figure 4B shows a closed plot where DNA concentration ranges from 0 to 80 fg/μL. The limit of detection (LOD) was estimated as the first concentration where the value was over the averaged fluorescence intensity plus 3S.D. for NC. The LOD for matched DNA was 7.37 fg/μL, corresponding to 2.36 amol per well. Based on the initial concentration of the target DNA solution, an estimated amount of target DNA was approximately 2.36 amol in the homemade microwell (the volume ∼7.07 μL, 3 mm diameter and 1 mm thickness) and 2.36 ymol in the photometric area (assuming the volume of the LIA, 30 μm diameter and 10 μm thickness). Therefore, we estimated the number of target DNA molecules captured in the LIA would be between 2.36 ymol (in minimum by assuming no DNA condensation at LIA) and 2.36 amol (in maximum by assuming all molecules in the microwell condensed in the LIA). Based on the LOD, the sensitivity of our PCR-free optical condensation technique with only 5 min of laser irradiation was one-order magnitude higher than that for digital PCR (∼200 fg/μL LOD).

Conclusions

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This study applies the “Light-induced Acceleration of Molecular Recognition” strategy to directly generate DNA-modified dissipative structures with heterogeneous probe particles (PSMPs and AuNPs) at the solid–liquid interface with enhanced optical force and photothermal effects without any genetic amplification step (PCR-free). This platform was DNA sequence-dependent and effectively distinguished one-base-pair mutations with a LOD (7.37 fg/μL) one-order magnitude higher than that for digital PCR, with only a few minutes of laser irradiation. The optically assembled microparticles and nanoparticles contained target dye-modified DNA with an estimated amount between 2.36 ymol (2.36 × 10–24 mol) and 2.36 amol (2.36 × 10–18 mol). This optical condensation-based technique can be used for rapid, highly sensitive, and sequence-specific DNA detection and provides a basis for the high-throughput quantitative measurement of cfDNA, mutation detection in ctDNA, and early diagnosis of diseases with higher specificity when combined with suitable biomarkers. The simplicity, rapid turnover, sensitivity, and specificity of optical condensation with heterogeneous probes may be extendable to other essential molecular recognition processes in addition to DNA hybridization, such as antigen–antibody reactions, sugar-lectin binding, and cell control on small biochips. The underlying mechanism would be applied to elucidate SNPs in DNA, such as KRAS and EGFR, (9,10) and the quantum probabilistic process of gene mutation. (11) Overall, our findings will inform the development of a portable platform for detecting environmental DNA in the fields of wildlife conservation and ecosystem management and promote a paradigm shift in liquid biopsies for rapid diagnostics and precision medicine.

Methods

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Materials

All the chemicals used in this study were of reagent grade. Chloroauric acid (HAuCl4, product # 073–00933), trisodium citrate dihydrate (product # 199–01781), and NaCl (product # 191–01665) were purchased from Fujifilm Wako Pure Chemical Industries, Ltd., and were used without any purification. Artificially synthesized oligonucleotides, including 5′-Alexa Fluor 488-capped DNA, 3′- and 5′-terminally thiolated DNA, and 3′- and 5′-terminally biotinylated DNA, were obtained from Thermo Fisher Scientific, and were used without any purification. The used sequences are represented in Table S1. Streptavidin-coated polystyrene microparticle (SA-PSMP) with a diameter of 2 μm (Cat # 24160–1) was purchased from Polysciences Inc. and used without any purification. Coverglass (22 × 32 mm, thickness 0.13–0.17 mm, product # C022321) was purchased from Matsunami Glass Ind., Ltd. Milli-Q grade (>18 MΩ) water with ultraviolet sterilization was used after filtering with 0.2 μm pore size throughout the experiment.

Preparation of Gold Nanoparticles

AuNPs with an average diameter of 30 nm and a typical concentration of 5.18 × 1011 particles/mL were prepared by the citrate reduction of HAuCl4. An aqueous solution of trisodium citrate dihydrate (2 wt %, 561 μL) was brought to 25 mL of MQ grade water with a stirrer bar, and heated to 80 °C with stirring (1500 rpm) in a water bath. An aqueous solution of HAuCl4 (1 wt %, 750 μL) was quickly added to the heated trisodium citrate solution. The stirring was continued for 20 min at 80 °C, then the solution was removed from the water bath. The solution was allowed to cool to room temperature. The resulting solution was characterized by measuring the UV–vis absorption spectrum with a spectrophotometer (UV-630BIO, JASCO), typically showing an absorption maximum at 526 nm (See a typical absorption spectrum in Figure S7). The derived AuNP dispersion liquid was stored in a glass bottle maintained at 25 °C before the modification of an oligonucleotide.

Modification of Probe DNA on the Probe Particles

For the modification of oligonucleotide to AuNPs, (33,35,38,39) 10 μL of 361 μM thiolated oligonucleotide aqueous solution (3′- or 5′-terminally thiolated DNA) was added in 850 μL of a 30 nm AuNP dispersion liquid, and incubated at 25 °C for 24 h. Thereafter, 40 μL of 2.5 M NaCl aqueous solution and 100 μL of 100 mM phosphate buffer (pH = 7.0, product # 168–27155) were added, resulting in 10 mM of phosphate buffer (pH 7.0) and 0.1 M of NaCl. The resulting dispersion liquid was maintained at 25 °C for 40 h, followed by centrifugation (SORVALL Biofuge primo R, Thermo Scientific) for 25 min at 15,000 rpm at ∼5 °C to remove any unreacted oligonucleotide. After the removal of the 950 μL supernatant, the AuNPs were washed with 10 mM phosphate buffer solution containing 0.1 M NaCl. After another round of centrifugation under the same conditions, the precipitate was dispersed in 10 mM phosphate buffer containing 0.3 M NaCl. The AuNPs modified with the 3′- and 5′-terminally thiolated DNA were denoted as Probe <I> and Probe <II>, respectively, as described in Table S1. The surface density of the DNA on a probe AuNP was estimated as 48 pmol/cm2. (39)
For the modification of oligonucleotide to SA-PSMPs, 10 μL of 361 μM biotinylated oligonucleotide aqueous solution (3′- or 5′-terminally biotinylated DNA) was added in 470 μL of a 2 μm SA-PSMP dispersion liquid (after 3times washed and 6 times dilution of the original SA-PSMP dispersion liquid with 10 mM phosphate buffer containing 0.1 M NaCl and 10 mg/mL Bovin Serum Albumin), and incubated at 4 °C for 1 h, followed by centrifugation (MCF-1350, LMS) for 3 min at 7.5 kG at ∼25 °C to remove any unreacted oligonucleotide. After the removal of the 456 μL supernatant, the SA-PSMPs were washed with 10 mM phosphate buffer solution containing 0.1 M NaCl. After another round of centrifugation under the same conditions, the precipitate was dispersed in 10 mM phosphate buffer containing 0.3 M NaCl. The SA-PSMPs modified with the 3′- and 5′-terminally biotinylated DNA were denoted as Probe <III> and Probe <IV>, respectively, as described in Table S1. The surface density of the probe DNA on a probe PSMP was estimated as 1.21 pmol/cm2. For the estimation, UV–vis absorption spectrum measurements by a spectrometer (V-630BIO, JASCO Corporation) were performed, where three dispersion liquid samples were measured; a) supernatant from the probe DNA-modified PSMP dispersion liquid, b) probe DNA solution, c) supernatant from a SA-PSMP dispersion liquid (6 times diluted from the original). The spectrum c was measured for removing any unwanted additions in spectrum a (for example, surfactants, stabilizers, etc. that are contained in the original SA-PSMP dispersion liquid, and phosphate buffer). Figure S8A shows the obtained three spectra, and Figure S8B shows the difference spectrum (a–c) and spectrum b. An absorbance reduction by the Streptavidin–Biotin binding on the SA-PSMPs was observed in Figure S8B. Absorbance at 260 nm (A260) of (a - c) and b were determined as 0.387 and 0.396, respectively. By considering the initial concentration of the probe DNA (CDNA = 3.61 μM), the solution volume (V = 1000 μL), the concentration (CMP = 5.401 × 108 particles/mL) and the surface area of the SA-PSMP (SMP = 12.566 μm2), the surface density of the probe DNA on a probe PSMP was calculated as followed,
Surfacedensity=(1−A260(a−c)A260(b))×CDNA×VCMP×SMP=(1−0.3870.396)×3.61⁡[μM]×1000⁡[μL]5.401×108⁡[particles/mL]×12.566⁡[μm2]=1.21⁡[pmol/cm2]

Light-Induced Acceleration and Fluorescence Imaging

The experimental setup used in this study is schematically described in Figure S9. The continuous infrared laser (ASF1JE01, 1064 nm, Furukawa Electric Co., Ltd.) for optical condensation was guided to an inverted optical microscope (Eclipse Ti–U, Nikon) using a backport adapter (LMSAD-NI-BP; Sigma-Koki). The condensation laser was focused with a 40 × objective lens (CFI S Plan Fluor ELWD 40XC NA = 0.6, Nikon). The input laser power was kept to 640 mW measured after the objective and a coverglass. Ten μL of sample-dispersed mixtures (each 5.0 μL of probe dispersion liquids, for example, 5 μL of Probe <II> and 5 μL of Probe <III>, and 5.0 μL of target DNA in 10 mM phosphate buffer (pH = 7.0)) was encapsulated in a homemade microwell (Figure S10). The homemade microwell consists of two coverglasses and a 1 mm thick spacer (KTD-12, 3 M Japan). A hole was made in the spacer by a cylindrical punch (ϕ ∼ 3 mm). The volume of the homemade microwell is estimated at about 7.07 μL. An optical transmission image of the top coverglass/liquid interface was recorded under bright field conditions using a Complementary Metal-Oxide-Semiconductor (CMOS) camera (DS-Fi3, 2880 × 2048 pixels, Nikon). Then, the 1064 nm laser was irradiated for 5 min, where the laser focus was set to 30 μm above the top coverglass/liquid interface. After the laser irradiation, a transmission image at the top coverglass/liquid interface was recorded again. Thereafter a fluorescence image was also recorded with an excitation light source Hg lamp and a filter set (FITC-A-Basic-NTE, Chroma). The recorded fluorescence images were analyzed with an image analysis software (NIS element, Nikon). Briefly, averaged brightness at the light-induced assembly (LIA) area (40 × 40 pixels at the weight center of LIA) and the background (BG) area (200 × 2048 pixels from the left edge of the image) were determined, and the fluorescence intensity was calculated as the difference of between the averaged brightness at LIA and BG.

Data Availability

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All data are presented in the paper and/or Supporting Information. Additional data may be requested from the corresponding authors.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssensors.4c02119.

  • Time course of transmission images with the heterogeneous probe-particles and target DNA without laser irradiation, fluorescence intensity for three experimental conditions, optical transmission image of the photothermal effect, theoretically calculated optical absorption of gold nanoparticles, stability, and functionality of probes, confirmation of the photobleaching effect of target DNA, experimentally observed absorption spectrum of AuNPs, surface density of probe DNA, schematic illustrations of experimental setup, DNA sequences, and explanation of the movie (PDF)

  • Movie of the optical transmission during optical condensation (MP4)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Authors
    • Shiho Tokonami - Research Institute for Light-induced Acceleration System (RILACS), Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan;  Department of Materials Science, Graduate School of Engineering, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan;  Email: [email protected]
    • Takuya Iida - Research Institute for Light-induced Acceleration System (RILACS), Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan;  Department of Physics, Graduate School of Science, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan;  Orcidhttps://orcid.org/0000-0003-1313-7025;  Email: [email protected]
  • Authors
    • Shuichi Toyouchi - Research Institute for Light-induced Acceleration System (RILACS), Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan;  Department of Physics, Graduate School of Science, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan;  Orcidhttps://orcid.org/0000-0002-4771-8015
    • Seiya Oomachi - Research Institute for Light-induced Acceleration System (RILACS), Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan;  Department of Physics, Graduate School of Science, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan;  Department of Materials Science, Graduate School of Engineering, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
    • Ryoma Hasegawa - Research Institute for Light-induced Acceleration System (RILACS), Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan;  Department of Physics, Graduate School of Science, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan;  Department of Materials Science, Graduate School of Engineering, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
    • Kota Hayashi - Research Institute for Light-induced Acceleration System (RILACS), Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan;  Department of Physics, Graduate School of Science, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan;  Department of Materials Science, Graduate School of Engineering, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
    • Yumiko Takagi - Research Institute for Light-induced Acceleration System (RILACS), Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan;  Department of Physics, Graduate School of Science, Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan
    • Mamoru Tamura - Research Institute for Light-induced Acceleration System (RILACS), Osaka Metropolitan University, 1-2 Gakuencho, Nakaku, Sakai, Osaka 599-8570, Japan;  Department of Materials Engineering Science, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan
  • Author Contributions

    TI and STok initiated the study and contributed equally to its design. SToy, SO, RH, KH, YT, STok, and TI performed the experiments on the light-induced acceleration of DNA hybridization with heterogeneous probe particles. MT, SToy, and TI performed the theoretical calculations. SToy, TI, and STok prepared the figures and manuscript. All authors critically reviewed and contributed to the manuscript. Conceptualization: TI, STok. Methodology: SToy, SO, RH, KH, YT, MT, STok, TI. Investigation: SToy, SO, RH, KH, YT, MT, STok, TI. Visualization: SToy, TI, STok. Funding acquisition: TI, STok, SToy, MT, KH. Project administration: TI, STok. Supervision: TI, STok. Writing–original draft: SToy, TI. Writing–review and editing: SToy, TI, STok.

  • Funding

    This work was partly supported by the JST Mirai Program (No. JPMJMI18GA, No. JPMJMI21G1, TI), Grant-in-Aid for Scientific Research (A) (No. JP17H00856, No. JP21H04964, TI; No. JP24H00433, STok), JST FOREST Program (No. JPMJFR201O, STok), Grant-in-Aid for Scientific Research on Innovative Areas (No. JP16H06507, TI), Grant-in-Aid for Research Activity Start-up (No. JP22K20512, SToy, and No. JP24K23034, KH), Grant-in-Aid for Early-Career Scientists (No. 20K15196, MT), Grant-in-Aid for JSPS Research Fellows (No. 21J21304, KH), Grant-in-Aid for Scientific Research (C) (No. JP24K08282, MT), Grant-in-Aid for Transformative Research Areas (A) (No. JP23H04594, MT) from the Japan Society for the Promotion of Science (JSPS) KAKENHI, and Key Project Grant Program of Osaka Prefecture University.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We would like to thank Prof. I. Nakase, Prof. A. Taguchi, and Prof. T. Satoh for their useful and fruitful discussions from the biological viewpoints.

References

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    A review. Abstr.: Over the past decade, various liq. biopsy techniques have emerged as viable alternatives to the anal. of traditional tissue biopsy samples. Such surrogate biopsies offer numerous advantages, including the relative ease of obtaining serial samples and overcoming the issues of interpreting one or more small tissue samples that might not reflect the entire tumor burden. To date, the majority of research in the area of liq. biopsies has focused on blood-based biomarkers, predominantly using plasma-derived circulating tumor DNA (ctDNA). However, ctDNA can also be obtained from various non-blood sources and these might offer unique advantages over plasma ctDNA. In this Review, we discuss advances in the anal. of ctDNA from non-blood sources, focusing on urine, cerebrospinal fluid, and pleural or peritoneal fluid, but also consider other sources of ctDNA. We discuss how these alternative sources can have a distinct yet complementary role to that of blood ctDNA anal. and consider various tech. aspects of non-blood ctDNA assay development. We also reflect on the settings in which non-blood ctDNA can offer distinct advantages over plasma ctDNA and explore some of the challenges assocd. with translating these alternative assays from academia into clin. use.
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  17. 17
    Sanger, F.; Nicklen, S.; Coulson, A. R. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U. S. A. 1977, 74, 5463– 5467,  DOI: 10.1073/pnas.74.12.5463
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    DNA sequencing with chain-terminating inhibitors
    Sanger, F.; Nicklen, S.; Coulson, A. R.
    Proceedings of the National Academy of Sciences of the United States of America (1977), 74 (12), 5463-7CODEN: PNASA6; ISSN:0027-8424.
    A new method for detg. nucleotide sequences in DNA is described. It is similar to the plus and minus method (Sanger, F., Coulson, A. R., 1975) but makes use of the 2',3'-dideoxy and arabinonucleoside analogs of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique was applied to the DNA of bacteriophage φX174 and was more rapid and more accurate than either the plus or the minus method.
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  18. 18
    Saiki, R. K.; Gelfand, D. H.; Stoffel, S.; Scharf, S. J.; Higuchi, R.; Horn, G. T.; Mullis, K. B.; Erlich, H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988, 239, 487– 491,  DOI: 10.1126/science.2448875
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    Primer-directed enzymic amplification of DNA with a thermostable DNA polymerase
    Saiki, Randall K.; Gelfand, David H.; Stoffel, Susanne; Scharf, Stephen J.; Higuchi, Russell; Horn, Glenn T.; Mullis, Kary B.; Erlich, Henry A.
    Science (Washington, DC, United States) (1988), 239 (4839), 487-91CODEN: SCIEAS; ISSN:0036-8075.
    A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure, enables amplification at higher temps., and significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by >10 million-fold with very high specificity, and DNA segments ≤2000 base pairs were readily amplified. The method was used to amplify and detected a target DNA mol. present once in a sample of 105 cells.
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    Rothberg, J. M.; Leamon, J. H. The development and impact of 454 sequencing. Nat. Biotechnol. 2008, 26, 1117– 1124,  DOI: 10.1038/nbt1485
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    Ashkin, A.; Dziedzic, J. M.; Yamane, T. Optical trapping and manipulation of single cells using infrared laser beams. Nature 1987, 330, 769– 771,  DOI: 10.1038/330769a0
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    Optical trapping and manipulation of single cells using infrared laser beams
    Ashkin A; Dziedzic J M; Yamane T
    Nature (1987), 330 (6150), 769-71 ISSN:0028-0836.
    Use of optical traps for the manipulation of biological particles was recently proposed, and initial observations of laser trapping of bacteria and viruses with visible argon-laser light were reported. We report here the use of infrared (IR) light to make much improved laser traps with significantly less optical damage to a variety of living cells. Using IR light we have observed the reproduction of Escherichia coli within optical traps at power levels sufficient to give manipulation at velocities up to approximately 500 micron s-1. Reproduction of yeast cells by budding was also achieved in IR traps capable of manipulating individual cells and clumps of cells at velocities of approximately micron s-1. Damage-free trapping and manipulation of suspensions of red blood cells of humans and of organelles located within individual living cells of spirogyra was also achieved, largely as a result of the reduced absorption of haemoglobin and chlorophyll in the IR. Trapping of many types of small protozoa and manipulation of organelles within protozoa is also possible. The manipulative capabilities of optical techniques were exploited in experiments showing separation of individual bacteria from one sample and their introduction into another sample. Optical orientation of individual bacterial cells in space was also achieved using a pair of laser-beam traps. These new manipulative techniques using IR light are capable of producing large forces under damage-free conditions and improve the prospects for wider use of optical manipulation techniques in microbiology.
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  21. 21
    Perkins, T. T.; Quake, S. R.; Smith, D. E.; Chu, S. Relaxation of a single DNA molecule observed by optical microscopy. Science 1994, 264, 822– 826,  DOI: 10.1126/science.8171336
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    Relaxation of a single DNA molecule observed by optical microscopy
    Perkins, Thomas T.; Quake, Stephen R.; Smith, Douglas E.; Chu, Steven
    Science (Washington, DC, United States) (1994), 264 (5160), 822-6CODEN: SCIEAS; ISSN:0036-8075.
    Single mols. of DNA, visualized in video fluorescence microscopy, were stretched to full extension in a flow, and their relaxation was measured when the flow stopped. The mols., attached by one end to a 1-μm bead, were manipulated in an aq. soln. with optical tweezers. Inverse Laplace transformations of the relaxation data yielded spectra of decaying exponentials with distinct peaks, and the longest time component (τ) increased with length (L) as τ ∼ L1.66±0.10. A rescaling anal. showed that most of the relaxation curves had a universal shape and their characteristic times (λt) increased as λt ∼ L1.65±0.13. These results are in qual. agreement with the theor. prediction of dynamic scaling.
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    Ishihara, H. Optical manipulation of nanoscale materials by linear and nonlinear resonant optical responses. Adv. Phys. X 2021,  DOI: 10.1080/23746149.2021.1885991
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    Single Organic Nanoparticles; Masuhara, H.; Nakanishi, H.; Sasaki, K., Eds.; Springer: Berlin, Germany, 2003.
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    Norregaard, K.; Metzler, R.; Ritter, C. M.; Berg-Sørensen, K.; Oddershede, L. B. Manipulation and Motion of Organelles and Single Molecules in Living Cells. Chem. Rev. 2017, 117, 4342– 4375,  DOI: 10.1021/acs.chemrev.6b00638
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    Manipulation and Motion of Organelles and Single Molecules in Living Cells
    Norregaard, Kamilla; Metzler, Ralf; Ritter, Christine M.; Berg-Soerensen, Kirstine; Oddershede, Lene B.
    Chemical Reviews (Washington, DC, United States) (2017), 117 (5), 4342-4375CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. The biomol. is among the most important building blocks of biol. systems, and a full understanding of its function forms the scaffold for describing the mechanisms of higher order structures as organelles and cells. Force is a fundamental regulatory mechanism of biomol. interactions driving many cellular processes. The forces on a mol. scale are exactly in the range that can be manipulated and probed with single mol. force spectroscopy. The natural environment of a biomol. is inside a living cell, hence, this is the most relevant environment for probing their function. In vivo studies are, however, challenged by the complexity of the cell. In this review, we start with presenting relevant theor. tools for analyzing single mol. data obtained in intracellular environments followed by a description of state-of-the art visualization techniques. The most commonly used force spectroscopy techniques, namely optical tweezers, magnetic tweezers, and at. force microscopy, are described in detail, and their strength and limitations related to in vivo expts. are discussed. Finally, recent exciting discoveries within the field of in vivo manipulation and dynamics of single mol. and organelles are reviewed.
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    Nishimura, Y.; Nishida, K.; Yamamoto, Y.; Ito, S.; Tokonami, S.; Iida, T. Control of submillimeter phase transition by collective photothermal effect. J. Phys. Chem. C 2014, 118, 18799– 18804,  DOI: 10.1021/jp506405w
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    Control of Submillimeter Phase Transition by Collective Photothermal Effect
    Nishimura, Yushi; Nishida, Keisuke; Yamamoto, Yojiro; Ito, Syoji; Tokonami, Shiho; Iida, Takuya
    Journal of Physical Chemistry C (2014), 118 (32), 18799-18804CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
    Local mol. states and biol. materials in small spaces ranging from the microscale to nanoscale can be modulated for medical and biol. applications using the photothermal effect (PTE). However, there have been only a few reports on exploiting the collective phenomena of localized surface plasmons (LSPs) to increase the amt. of light-induced heat for the control of material states and the generation of macroscopic assembled structures. Here, we clarify that microbeads covered with a vast no. of Ag nanoparticles can induce a large PTE and generate a submillimeter bubble within several tens of seconds under the synergetic effect of the light-induced force (LIF) and photothermal convection enhanced by collective phenomena of LSPs. Control of the phase transition induced by such a "collective photothermal effect" enables rapid assembling of macroscopic structures consisting of nanomaterials, which would be used for detection of a small amt. of proteins based on light-induced heat coagulation.
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    Iida, T. Development of Innovative Bio-measurement Technology by Micro-flow Light-Induced Acceleration; Horiba Tech Rep; 2021; Vol. 55, p 11.
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    Tokonami, S.; Iida, T. Review: Novel sensing strategies for bacterial detection based on active and passive methods driven by external field. Anal. Chim. Acta 2017, 988, 1– 16,  DOI: 10.1016/j.aca.2017.07.034
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    Tokonami, S.; Kurita, S.; Yoshikawa, R.; Sakurai, K.; Suehiro, T.; Yamamoto, Y.; Tamura, M.; Karthaus, O.; Iida, T. Light-induced assembly of living bacteria with honeycomb substrate. Sci. Adv. 2020, 6, eaaz5757  DOI: 10.1126/sciadv.aaz5757
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    Iida, T.; Hamatani, S.; Takagi, Y.; Fujiwara, K.; Tamura, M.; Tokonami, S. Attogram-level light-induced antigen-antibody binding confined in microflow. Commun. Biol. 2022, 5, 1053,  DOI: 10.1038/s42003-022-03946-0
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    Fujiwara, K.; Takagi, Y.; Tamura, M.; Omura, M.; Morimoto, K.; Nakase, I.; Tokonami, S.; Iida, T. Ultrafast sensitivity-controlled and specific detection of extracellular vesicles using optical force with antibody-modified microparticles in a microflow system. Nanoscale Horiz. 2023, 8, 1034,  DOI: 10.1039/D2NH00576J
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    Kanoda, M.; Hayashi, K.; Takagi, Y.; Tamura, M.; Tokonami, S.; Iida, T. High-throughput light-induced immunoassay with milliwatt-level laser under one-minute optical antibody-coating on nanoparticle-imprinted substrate. npj Biosensing 2024, 1, 1,  DOI: 10.1038/s44328-024-00004-z
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    Nakase, I.; Miyai, M.; Noguchi, K.; Tamura, M.; Yamamoto, Y.; Nishimura, Y.; Omura, M.; Hayashi, K.; Futaki, S.; Tokonami, S.; Iida, T. Light-Induced Condensation of Biofunctional Molecules around Targeted Living Cells to Accelerate Cytosolic Delivery. Nano Lett. 2022, 22, 9805– 9814,  DOI: 10.1021/acs.nanolett.2c02437
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    Light-Induced Condensation of Biofunctional Molecules around Targeted Living Cells to Accelerate Cytosolic Delivery
    Nakase, Ikuhiko; Miyai, Moe; Noguchi, Kosuke; Tamura, Mamoru; Yamamoto, Yasuyuki; Nishimura, Yushi; Omura, Mika; Hayashi, Kota; Futaki, Shiroh; Tokonami, Shiho; Iida, Takuya
    Nano Letters (2022), 22 (24), 9805-9814CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    The light-induced force and convection can be enhanced by the collective effect of electrons (superradiance and red shift) in high-d. metallic nanoparticles, leading to macroscopic assembly of target mols. We here demonstrate application of the light-induced assembly for drug delivery system with enhancement of cell membrane accumulation and penetration of biofunctional mols. including cell-penetrating peptides (CPPs) with superradiance-mediated photothermal convection. For induction of photothermal assembly around targeted living cells in cell culture medium, IR continuous-wave laser light was focused onto high-d. gold-particle-bound glass bottom dishes exhibiting plasmonic superradiance or thin gold-film-coated glass bottom dishes. In this system, the biofunctional mols. can be concd. around the targeted living cells and internalized into them only by 100 s laser irradn. Using this simple approach, we successfully achieved enhanced cytosolic release of the CPPs and apoptosis induction using a pro-apoptotic domain with a very low peptide concn. (nM level) by light-induced condensation.
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    Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature 1996, 382, 607– 609,  DOI: 10.1038/382607a0
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    A DNA-based method for rationally assembling nanoparticles into macroscopic materials
    Mirkin, Chad A.; Letsinger, Robert L.; Mucic, Robert C.; Storhoff, James J.
    Nature (London) (1996), 382 (6592), 607-609CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)
    Colloidal particles of metals and semiconductors have potentially useful optical, optoelectronic and material properties that derive from their small (nanoscopic) size. These properties might lead to applications including chem. sensors, spectroscopic enhancers, quantum dot and nanostructure fabrication, and microimaging methods. A great deal of control can now be exercised over the chem. compn., size and polydispersity of colloidal particles, and many methods have been developed for assembling them into useful aggregates and materials. Here we describe a method for assembling colloidal gold nanoparticles rationally and reversibly into macroscopic aggregates. The method involves attaching to the surfaces of two batches of 13-nm gold particles non-complementary DNA oligonucleotides capped with thiol groups, which bind to gold. When we add to the soln. an oligonucleotide duplex with 'sticky ends' that are complementary to the two grafted sequences, the nanoparticles self-assemble into aggregates. This assembly process can be reversed by thermal denaturation. This strategy should now make it possible to tailor the optical, electronic and structural properties of the colloidal aggregates by using the specificity of DNA interactions to direct the interactions between particles of different size and compn.
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    Alivisatos, A. P.; Johnsson, K. P.; Peng, X.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P.; Schultz, P. G. Organization of ’nanocrystal molecules’ using DNA. Nature 1996, 382, 609– 611,  DOI: 10.1038/382609a0
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    Organization of 'nanocrystal molecules' using DNA
    Alivisatos, A. Paul; Johnsson, Kai P.; Peng, Xiaogang; Wilson, Troy E.; Loweth, Colin J.; Bruchez, Marcel P., Jr.; Schultz, Peter G.
    Nature (London) (1996), 382 (6592), 609-611CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)
    The authors describe a strategy for the synthesis of 'nanocrystal mols.', in which discrete nos. of Au nanocrystals are organized into spatially defined structures based on Watson-Crick base-pairing interactions. The authors attach single-stranded DNA oligonucleotides of defined length and sequence to individual nanocrystals, and these assemble into dimers and trimers on addn. of a complementary single-stranded DNA template. The authors anticipate that this approach should allow the construction of more complex two- and three-dimensional assemblies.
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    Pinheiro, A.; Han, D.; Shih, W.; Yan, H. Challenges and opportunities for structural DNA nanotechnology. Nat. Nanotechnol. 2011, 6, 763– 772,  DOI: 10.1038/nnano.2011.187
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    Challenges and opportunities for structural DNA nanotechnology
    Pinheiro, Andre V.; Han, Dongran; Shih, William M.; Yan, Hao
    Nature Nanotechnology (2011), 6 (12), 763-772CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
    A review. DNA mols. have been used to build a variety of nanoscale structures and devices over the past 30 years, and potential applications have begun to emerge. But the development of more advanced structures and applications will require a no. of issues to be addressed, the most significant of which are the high cost of DNA and the high error rate of self-assembly. Here we examine the tech. challenges in the field of structural DNA nanotechnol. and outline some of the promising applications that could be developed if these hurdles can be overcome. In particular, we highlight the potential use of DNA nanostructures in mol. and cellular biophysics, as biomimetic systems, in energy transfer and photonics, and in diagnostics and therapeutics for human health.
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    Mu, B.; Zhang, J.; McNicholas, T. P.; Reuel, N. F.; Kruss, S.; Strano, M. S. Recent advances in molecular recognition based on nanoengineered platforms. Acc. Chem. Res. 2014, 47, 979– 988,  DOI: 10.1021/ar400162w
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    Recent Advances in Molecular Recognition Based on Nanoengineered Platforms
    Mu, Bin; Zhang, Jingqing; McNicholas, Thomas P.; Reuel, Nigel F.; Kruss, Sebastian; Strano, Michael S.
    Accounts of Chemical Research (2014), 47 (4), 979-988CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review. Nanoparticles and nanoengineered platforms have great potential for technologies involving biomoleuclar detection or cell-related biosensing, and have provided effective chem. interfaces for mol. recognition. Typically, chemists work on the modification of synthetic polymers or macromols., which they link to the nanoparticles by covalent or noncovalent approaches. The motivation for chem. modification is to enhance the selectivity and sensitivity, and to improve the biocompatibility for the in vivo applications. In this Account, we present recent advances in the development and application of chem. interfaces for mol. recognition for nanoparticles and nanoengineered platforms, in particular single-walled carbon nanotubes (SWNTs). We discuss emerging approaches for recognizing small mols., glycosylated proteins, and serum biomarkers. For example, we compare and discuss detection methods for ATP, NO, H2O2, and monosaccharides for recent nanomaterials. Fluorometric detection appears to have great potential for quantifying concn. gradients and detg. their location in living cells. For macromol. detection, new methods for glycoprofiling using such interfaces appear promising, and benefit specifically from the potential elimination of cumbersome labeling and liberation steps during conventional anal. of glycans, augmenting the currently used mass spectrometry (MS), capillary electrophoresis (CE), and liq. chromatog. (LC) methods. In particular, we demonstrated the great potential of fluorescent SWNTs for glycan-lectin interactions sensing. In this case, SWNTs are noncovalently functionalized to introduce a chelated nickel group. This group provides a docking site for the His-tagged lectin and acts as the signal modulator. As the nickel proximity to the SWNT surface changes, the fluorescent signal is increased or attenuated. When a free glycan or glycosylated probe interacts with the lectin, the signal increases and they are able to obtain loading curves similar to surface plasmon resonance measurements. They demonstrate the sensitivity and specificity of this platform with two higher-affined glycan-lectin pairs: fucose (Fuc) to PA-IIL and N-acetylglucosamine (GlcNAc) to GafD. Lastly, we discuss how developments in protein biomarker detection in general are benefiting specifically from label-free mol. recognition. Elec. field effect transistors, chemi-resistive and fluorometric nanosensors based on various nanomaterials have demonstrated substantial progress in recent years in addressing this challenging problem. In this Account, we compare the balance between sensitivity, selectivity, and nonspecific adsorption for various applications. In particular, our group has utilized SWNTs as fluorescence sensors for label-free protein-protein interaction measurements. In this assay, we have encapsulated each nanotube in a biocompatible polymer, chitosan, which has been further modified to conjugate nitrilotriacetic acid (NTA) groups. After Ni2+ chelation, NTA Ni2+ complexes bind to his-tagged proteins, resulting in a local environment change of the SWNT array, leading to optical fluorescence modulation with detection limit down to 100 nM. We have further engineered the platform to monitor single protein binding events, with an even lower detection limit down to 10 pM.
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    Seaberg, J.; Montazerian, H.; Hossen, M. N.; Bhattacharya, R.; Khademhosseini, A.; Mukherjee, P. Hybrid nanosystems for biomedical applications. ACS Nano 2021, 15 (2), 2099– 2142,  DOI: 10.1021/acsnano.0c09382
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    Hybrid Nanosystems for Biomedical Applications
    Seaberg, Joshua; Montazerian, Hossein; Hossen, Md Nazir; Bhattacharya, Resham; Khademhosseini, Ali; Mukherjee, Priyabrata
    ACS Nano (2021), 15 (2), 2099-2142CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    A review. Inorg./org. hybrid nanosystems have been increasingly developed for their versatility and efficacy at overcoming obstacles not readily surmounted by nonhybridized counterparts. Currently, hybrid nanosystems are implemented for gene therapy, drug delivery, and phototherapy in addn. to tissue regeneration, vaccines, antibacterials, biomol. detection, imaging probes, and theranostics. Though diverse, these nanosystems can be classified according to foundational inorg./org. components, accessory moieties, and architecture of hybridization. Within this Review, we begin by providing a historical context for the development of biomedical hybrid nanosystems before describing the properties, synthesis, and characterization of their component building blocks. Afterward, we introduce the architectures of hybridization and highlight recent biomedical nanosystem developments by area of application, emphasizing hybrids of distinctive utility and innovation. Finally, we draw attention to ongoing clin. trials before recapping our discussion of hybrid nanosystems and providing a perspective on the future of the field.
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    Tokonami, S.; Shiigi, H.; Nagaoka, T. Open bridge-structured gold nanoparticle array for label-free DNA detection. Anal. Chem. 2008, 80, 8071– 8075,  DOI: 10.1021/ac801088u
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    Open Bridge-Structured Gold Nanoparticle Array for Label-Free DNA Detection
    Tokonami, Shiho; Shiigi, Hiroshi; Nagaoka, Tsutomu
    Analytical Chemistry (Washington, DC, United States) (2008), 80 (21), 8071-8075CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    We focused on changes in the elec. property of the open bridge-structured gold nanoparticles array consisting of 46-nm parent and 12-nm son gold nanoparticles by hybridization and applied it for a simple elec. DNA detection. Since a target DNA of a 24-mer oligonucleotide was added to the probe DNA modified 12-nm Au nanoparticles, which was arranged on the gap between the 46-nm Au particles, the response was read by an elec. readout system. Even in a simple measuring method, we obtained a rapid response to the cDNA with a high S/N ratio of 30 over a wide concn. range and a detection limit of 5.0 fmol. Moreover, the array discriminated 1-base mismatches, regardless of their location in the DNA sequence, which enabled us to detect single-nucleotide polymorphism, which is one of the important diagnoses, without any polymerase chain reaction amplification, sophisticated instrumentation, or fluorescent labeling through an easy-to-handle elec. readout system.
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    Iida, T.; Nishimura, Y.; Tamura, M.; Nishida, K.; Ito, S.; Tokonami, S. Submillimetre Network Formation by Light-induced Hybridization of Zeptomole-level DNA. Sci. Rep 2016, 6, 37768,  DOI: 10.1038/srep37768
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    39
    Submillimetre Network Formation by Light-induced Hybridization of Zeptomole-level DNA
    Iida, Takuya; Nishimura, Yushi; Tamura, Mamoru; Nishida, Keisuke; Ito, Syoji; Tokonami, Shiho
    Scientific Reports (2016), 6 (), 37768CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    Macroscopic unique self-assembled structures are produced via double-stranded DNA formation (hybridization) as a specific binding essential in biol. systems. However, a large amt. of complementary DNA mols. are usually required to form an optically observable structure via natural hybridization, and the detection of small amts. of DNA less than femtomole requires complex and time-consuming procedures. Here, we demonstrate the laser-induced acceleration of hybridization between zeptomole-level DNA and DNA-modified nanoparticles (NPs), resulting in the assembly of a submillimetre network-like structure at the desired position with a dramatic spectral modulation within several minutes. The gradual enhancement of light-induced force and convection facilitated the two-dimensional network growth near the air-liq. interface with optical and fluidic symmetry breakdown. The simultaneous microscope observation and local spectroscopy revealed that the assembling process and spectral change are sensitive to the DNA sequence. Our findings establish innovative guiding principles for facile bottom-up prodn. via various biomol. recognition events.
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    ACS Nano (2020), 14 (7), 8570-8583CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    Femtosecond (fs) laser pulsed excitation of plasmonic nanoparticle (NP)-biomol. conjugates is a promising method to locally heat biol. materials. Studies have demonstrated that fs pulses of light can modulate the activity of DNA or proteins when attached to plasmonic NPs; however, the precision over subsequent biol. function remains largely undetd. Specifically, the temp. the localized biomols. "experience" remains unknown. The authors used 55 nm gold nanoparticles (AuNPs) displaying double-stranded (ds) DNA to examine how, for dsDNA with different melting temps., the laser pulse energy fluence and bulk soln. temp. affect the rate of local DNA denaturation. A universal "template" single-stranded DNA was attached to the AuNP surface, and three dye-labeled probe strands, distinct in length and melting temp., were hybridized to it creating three individual dsDNA-AuNP bioconjugates. The dye-labeled probe strands were used to quantify the rate and amt. of DNA release after a given no. of light pulses, which was then correlated to the dsDNA denaturation temp., resulting in a quant. nanothermometer. The localized DNA denaturation rate could be modulated by more than threefold over the biol. relevant range of 8-53° by varying pulse energy fluence, DNA melting temp., and surrounding bath temp. With a modified dissocn. equation tailored for this system, a "sensed" temp. parameter was extd. and compared to simulated AuNP temp. profiles. Detg. actual biol. responses in such systems can allow researchers to design precision nanoscale photothermal heating sources.
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  • Abstract

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    Figure 1

    Figure 1. Light-induced acceleration of DNA hybridization with optically assembled DNA-functionalized gold nanoparticles (DNA-AuNP, Probe 1) and polystyrene microparticles (DNA-PSMP, Probe 2) as heterogeneous probe particles for PCR-free DNA detection. (A) Schematic illustration of experimental design. An infrared laser was loosely focused near the glass–liquid interface of a homemade microwell, where probe particles and fluorescent dye-labeled target DNA molecules were transported by light-induced force (LIF) and light-induced convection (LIC) flow and then assembled. Inside the light-induced assembly (LIA), the complementary target DNA molecules bridge the heterogeneous probes by forming double-stranded DNA. AuNPs facilitate sequence-specific DNA detection by enhancing the fluorescence signal of target DNA through surface plasmon resonance and increasing the temperature inside the LIA through excellent photothermal effects. (B) Transmission images recorded ‘a’ before laser irradiation, during (b; 0 s, c; 1 min, d; 2 min, e; 3 min, f, 4 min, g; 5 min), and after the optical condensation of heterogeneous probe particles and target DNA. Images b-g were recorded by intentionally shifting the focal plane above 30 μm from the coverglass–liquid interface to expand the laser spot about 30 μm. In h, the focal plane moved to the ceiling of the microwell after laser irradiation. Scale bar: 100 μm. See also Movie S1.

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    Figure 2

    Figure 2. Light-induced acceleration of DNA hybridization. (A) The LIA of target DNA and heterogeneous probe particle (top) was monitored by transmission imaging using a CMOS camera before (0 s, left) and after 1064 nm continuous wave laser irradiation for 300 s (middle). Fluorescence images were captured after laser irradiation (right). The target DNA was complementary to the probe DNA sequence (Matched, 7.37 pg/μL, upper row) or fully mismatched DNA (Mismatched, 7.37 pg/μL, middle row). A negative control (target DNA 0 pg/μL) was examined under the same experimental conditions. (B) The same light-induced acceleration experiment (as in A) performed with the homogeneous probes. Fluorescence intensity (Fluo. Int.) at the LIA and background (BG) as well as the difference (Diff) between them are listed on the right side.

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    Figure 3

    Figure 3. Dependence of optical acceleration on DNA sequence. (A) Relative fluorescence intensity (normalized to that obtained with Matched DNA) with heterogeneous probe particles (red circles) and homogeneous probe particles (blue triangles) with varying numbers of mismatching base pairs. The error bar represents the SD (n = 3). For 1-base and 2-base-mismatched DNA, two sequences of each condition (MM1a/MM1b, and MM2a/MM2b) were evaluated. MM1a and MM2a are represented as the opened marker, and MM1b and MM2b are represented as the closed marker. (B) Schematic illustration changes in DNA structure with melting temperature (Tm). The Tm for Matched DNA is generally higher than that for Mismatched DNA (Tm’). In optical condensation using heterogeneous probe particles, the temperature inside the LIA (TLIA) is expected to increase above room temperature owing to the photothermal effect of the AuNPs. See also the DNA sequences in Table S2.

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    Figure 4

    Figure 4. Calibration curve of DNA optical condensation with heterogeneous probe particles. (A) Fluorescence intensities obtained with Matched DNA (red circles) and Mismatched DNA (blue diamonds) were plotted as a function of DNA concentration. The error bars represent the SD (n = 3). (B) Closed plot of fluorescence intensity as a function of DNA concentrations ranging from 0 to 80 fg/μL. The dashed line represents the averaged fluorescence intensity +3S.D at the NC (DNA concentration 0 fg/μL).

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      Huang, L.; Guo, Z.; Wang, F.; Fu, L. KRAS mutation: from undruggable to druggable in cancer. Signal Transduct Target Ther 2021, 6, 386,  DOI: 10.1038/s41392-021-00780-4
      9
      KRAS mutation: from undruggable to druggable in cancer
      Huang Lamei; Guo Zhixing; Wang Fang; Fu Liwu
      Signal transduction and targeted therapy (2021), 6 (1), 386 ISSN:.
      Cancer is the leading cause of death worldwide, and its treatment and outcomes have been dramatically revolutionised by targeted therapies. As the most frequently mutated oncogene, Kirsten rat sarcoma viral oncogene homologue (KRAS) has attracted substantial attention. The understanding of KRAS is constantly being updated by numerous studies on KRAS in the initiation and progression of cancer diseases. However, KRAS has been deemed a challenging therapeutic target, even "undruggable", after drug-targeting efforts over the past four decades. Recently, there have been surprising advances in directly targeted drugs for KRAS, especially in KRAS (G12C) inhibitors, such as AMG510 (sotorasib) and MRTX849 (adagrasib), which have obtained encouraging results in clinical trials. Excitingly, AMG510 was the first drug-targeting KRAS (G12C) to be approved for clinical use this year. This review summarises the most recent understanding of fundamental aspects of KRAS, the relationship between the KRAS mutations and tumour immune evasion, and new progress in targeting KRAS, particularly KRAS (G12C). Moreover, the possible mechanisms of resistance to KRAS (G12C) inhibitors and possible combination therapies are summarised, with a view to providing the best regimen for individualised treatment with KRAS (G12C) inhibitors and achieving truly precise treatment.
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    10. 10
      Sharma, S. V.; Bell, D. W.; Settleman, J.; Haber, D. A. Epidermal growth factor receptor mutations in lung cancer. Nat. Rev. Cancer 2007, 7, 169– 181,  DOI: 10.1038/nrc2088
      10
      Epidermal growth factor receptor mutations in lung cancer
      Sharma, Sreenath V.; Bell, Daphne W.; Settleman, Jeffrey; Haber, Daniel A.
      Nature Reviews Cancer (2007), 7 (3), 169-181CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)
      A review. The development and clin. application of inhibitors that target the epidermal growth factor receptor (EGFR) provide important insights for new lung cancer therapies, as well as for the broader field of targeted cancer therapies. We review the results of genetic, biochem. and clin. studies focused on somatic mutations of EGFR that are assocd. with the phenomenon of oncogene addiction, describing 'oncogenic shock' as a mechanistic explanation for the apoptosis that follows the acute treatment of susceptible cells with kinase inhibitors. Understanding the genetic heterogeneity of epithelial tumors and devising strategies to circumvent their rapid acquisition of resistance to targeted kinase inhibitors are essential to the successful use of targeted therapies in common epithelial cancers.
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    11. 11
      Slocombe, L.; Sacchi, M.; Al-Khalili, J. An open quantum systems approach to proton tunnelling in DNA. Commun. Phys. 2022, 5, 109,  DOI: 10.1038/s42005-022-00881-8
      11
      An open quantum systems approach to proton tunnelling in DNA
      Slocombe, Louie; Sacchi, Marco; Al-Khalili, Jim
      Communications Physics (2022), 5 (1), 109CODEN: CPOHDJ; ISSN:2399-3650. (Nature Portfolio)
      One of the most important topics in mol. biol. is the genetic stability of DNA. One threat to this stability is proton transfer along the hydrogen bonds of DNA that could lead to tautomerisation, hence creating point mutations. We present a theor. anal. of the hydrogen bonds between the Guanine-Cytosine (G-C) nucleotide, which includes an accurate model of the structure of the base pairs, the quantum dynamics of the hydrogen bond proton, and the influence of the decoherent and dissipative cellular environment. We det. that the quantum tunnelling contribution to the proton transfer rate is several orders of magnitude larger than the classical over-the-barrier hopping. Due to the significance of the quantum tunnelling even at biol. temps., we find that the canonical and tautomeric forms of G-C inter-convert over timescales far shorter than biol. ones and hence thermal equil. is rapidly reached. Furthermore, we find a large tautomeric occupation probability of 1.73 x 10-4, suggesting that such proton transfer may well play a far more important role in DNA mutation than has hitherto been suggested. Our results could have far-reaching consequences for current models of genetic mutations.
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    12. 12
      Kumagai, T.; Kaizu, M.; Hatta, S.; Okuyama, H.; Aruga, T.; Hamada, I.; Morikawa, Y. Direct observation of hydrogen-bond exchange within a single water dimer. Phys. Rev. Lett. 2008, 100, 166101,  DOI: 10.1103/PhysRevLett.100.166101
      12
      Direct Observation of Hydrogen-Bond Exchange within a Single Water Dimer
      Kumagai, T.; Kaizu, M.; Hatta, S.; Okuyama, H.; Aruga, T.; Hamada, I.; Morikawa, Y.
      Physical Review Letters (2008), 100 (16), 166101/1-166101/4CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
      The dynamics of water dimers was investigated at the single-mol. level by using a scanning tunneling microscope. The two mols. in a water dimer, bound on a Cu(110) surface at 6 K, were obsd. to exchange their roles as hydrogen-bond donor and acceptor via hydrogen-bond rearrangement. The interchange rate is ∼60 times higher for (H2O)2 than for (D2O)2, suggesting that quantum tunneling is involved in the process. The interchange rate is enhanced upon excitation of the intermol. mode that correlates with the reaction coordinate.
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    13. 13
      Djurhuus, A.; Closek, C. J.; Kelly, R. P.; Pitz, K. J.; Michisaki, R. P.; Starks, H. A.; Walz, K. R.; Andruszkiewicz, E. A.; Olesin, E.; Hubbard, K.; Montes, E.; Otis, D.; Muller-Karger, F. E.; Chavez, F. P.; Boehm, A. B.; Breitbart, M. Environmental DNA reveals seasonal shifts and potential interactions in a marine community. Nat. Commun. 2020, 11, 254,  DOI: 10.1038/s41467-019-14105-1
      There is no corresponding record for this reference.
    14. 14
      Lo, D. Y. M.; Han, D. S. C.; Jiang, P.; Chiu, R. W. K. Epigenetics, fragmentomics, and topology of cell-freeDNA in liquid biopsies. Science 2021, 372, eaaw3616,  DOI: 10.1126/science.aaw3616
      There is no corresponding record for this reference.
    15. 15
      Zhou, Q.; Kang, G.; Jiang, P.; Qiao, R.; Lam, W. K. J.; Yu, S. C. Y.; Ma, M. L.; Ji, L.; Cheng, S. H.; Gai, W.; Peng, W.; Shang, H.; Chan, R. W. Y.; Chan, S. L.; Wong, G. L. H.; Hiraki, L. T.; Volpi, S.; Wong, V. W. S.; Wong, J.; Chiu, R. W. K.; Chan, K. C. A.; Lo, Y. M. D. Epigenetic analysis of cell-free DNA by fragmentomic profiling. Proc. Natl. Acad. Sci. U. S. A. 2022, 119, e2209852119  DOI: 10.1073/pnas.2209852119
      There is no corresponding record for this reference.
    16. 16
      Tivey, A.; Church, M.; Rothwell, D.; Dive, C.; Cook, N. Circulating tumour DNA ─ looking beyond the blood. Nat. Rev. Clin. Oncol. 2022, 19, 600,  DOI: 10.1038/s41571-022-00660-y
      16
      Circulating tumour DNA - looking beyond the blood
      Tivey, Ann; Church, Matt; Rothwell, Dominic; Dive, Caroline; Cook, Natalie
      Nature Reviews Clinical Oncology (2022), 19 (9), 600-612CODEN: NRCOAA; ISSN:1759-4774. (Nature Portfolio)
      A review. Abstr.: Over the past decade, various liq. biopsy techniques have emerged as viable alternatives to the anal. of traditional tissue biopsy samples. Such surrogate biopsies offer numerous advantages, including the relative ease of obtaining serial samples and overcoming the issues of interpreting one or more small tissue samples that might not reflect the entire tumor burden. To date, the majority of research in the area of liq. biopsies has focused on blood-based biomarkers, predominantly using plasma-derived circulating tumor DNA (ctDNA). However, ctDNA can also be obtained from various non-blood sources and these might offer unique advantages over plasma ctDNA. In this Review, we discuss advances in the anal. of ctDNA from non-blood sources, focusing on urine, cerebrospinal fluid, and pleural or peritoneal fluid, but also consider other sources of ctDNA. We discuss how these alternative sources can have a distinct yet complementary role to that of blood ctDNA anal. and consider various tech. aspects of non-blood ctDNA assay development. We also reflect on the settings in which non-blood ctDNA can offer distinct advantages over plasma ctDNA and explore some of the challenges assocd. with translating these alternative assays from academia into clin. use.
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    17. 17
      Sanger, F.; Nicklen, S.; Coulson, A. R. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U. S. A. 1977, 74, 5463– 5467,  DOI: 10.1073/pnas.74.12.5463
      17
      DNA sequencing with chain-terminating inhibitors
      Sanger, F.; Nicklen, S.; Coulson, A. R.
      Proceedings of the National Academy of Sciences of the United States of America (1977), 74 (12), 5463-7CODEN: PNASA6; ISSN:0027-8424.
      A new method for detg. nucleotide sequences in DNA is described. It is similar to the plus and minus method (Sanger, F., Coulson, A. R., 1975) but makes use of the 2',3'-dideoxy and arabinonucleoside analogs of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique was applied to the DNA of bacteriophage φX174 and was more rapid and more accurate than either the plus or the minus method.
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    18. 18
      Saiki, R. K.; Gelfand, D. H.; Stoffel, S.; Scharf, S. J.; Higuchi, R.; Horn, G. T.; Mullis, K. B.; Erlich, H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988, 239, 487– 491,  DOI: 10.1126/science.2448875
      18
      Primer-directed enzymic amplification of DNA with a thermostable DNA polymerase
      Saiki, Randall K.; Gelfand, David H.; Stoffel, Susanne; Scharf, Stephen J.; Higuchi, Russell; Horn, Glenn T.; Mullis, Kary B.; Erlich, Henry A.
      Science (Washington, DC, United States) (1988), 239 (4839), 487-91CODEN: SCIEAS; ISSN:0036-8075.
      A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure, enables amplification at higher temps., and significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by >10 million-fold with very high specificity, and DNA segments ≤2000 base pairs were readily amplified. The method was used to amplify and detected a target DNA mol. present once in a sample of 105 cells.
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    19. 19
      Rothberg, J. M.; Leamon, J. H. The development and impact of 454 sequencing. Nat. Biotechnol. 2008, 26, 1117– 1124,  DOI: 10.1038/nbt1485
      There is no corresponding record for this reference.
    20. 20
      Ashkin, A.; Dziedzic, J. M.; Yamane, T. Optical trapping and manipulation of single cells using infrared laser beams. Nature 1987, 330, 769– 771,  DOI: 10.1038/330769a0
      20
      Optical trapping and manipulation of single cells using infrared laser beams
      Ashkin A; Dziedzic J M; Yamane T
      Nature (1987), 330 (6150), 769-71 ISSN:0028-0836.
      Use of optical traps for the manipulation of biological particles was recently proposed, and initial observations of laser trapping of bacteria and viruses with visible argon-laser light were reported. We report here the use of infrared (IR) light to make much improved laser traps with significantly less optical damage to a variety of living cells. Using IR light we have observed the reproduction of Escherichia coli within optical traps at power levels sufficient to give manipulation at velocities up to approximately 500 micron s-1. Reproduction of yeast cells by budding was also achieved in IR traps capable of manipulating individual cells and clumps of cells at velocities of approximately micron s-1. Damage-free trapping and manipulation of suspensions of red blood cells of humans and of organelles located within individual living cells of spirogyra was also achieved, largely as a result of the reduced absorption of haemoglobin and chlorophyll in the IR. Trapping of many types of small protozoa and manipulation of organelles within protozoa is also possible. The manipulative capabilities of optical techniques were exploited in experiments showing separation of individual bacteria from one sample and their introduction into another sample. Optical orientation of individual bacterial cells in space was also achieved using a pair of laser-beam traps. These new manipulative techniques using IR light are capable of producing large forces under damage-free conditions and improve the prospects for wider use of optical manipulation techniques in microbiology.
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    21. 21
      Perkins, T. T.; Quake, S. R.; Smith, D. E.; Chu, S. Relaxation of a single DNA molecule observed by optical microscopy. Science 1994, 264, 822– 826,  DOI: 10.1126/science.8171336
      21
      Relaxation of a single DNA molecule observed by optical microscopy
      Perkins, Thomas T.; Quake, Stephen R.; Smith, Douglas E.; Chu, Steven
      Science (Washington, DC, United States) (1994), 264 (5160), 822-6CODEN: SCIEAS; ISSN:0036-8075.
      Single mols. of DNA, visualized in video fluorescence microscopy, were stretched to full extension in a flow, and their relaxation was measured when the flow stopped. The mols., attached by one end to a 1-μm bead, were manipulated in an aq. soln. with optical tweezers. Inverse Laplace transformations of the relaxation data yielded spectra of decaying exponentials with distinct peaks, and the longest time component (τ) increased with length (L) as τ ∼ L1.66±0.10. A rescaling anal. showed that most of the relaxation curves had a universal shape and their characteristic times (λt) increased as λt ∼ L1.65±0.13. These results are in qual. agreement with the theor. prediction of dynamic scaling.
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    22. 22
      Ishihara, H. Optical manipulation of nanoscale materials by linear and nonlinear resonant optical responses. Adv. Phys. X 2021,  DOI: 10.1080/23746149.2021.1885991
      There is no corresponding record for this reference.
    23. 23
      Single Organic Nanoparticles; Masuhara, H.; Nakanishi, H.; Sasaki, K., Eds.; Springer: Berlin, Germany, 2003.
      There is no corresponding record for this reference.
    24. 24
      Norregaard, K.; Metzler, R.; Ritter, C. M.; Berg-Sørensen, K.; Oddershede, L. B. Manipulation and Motion of Organelles and Single Molecules in Living Cells. Chem. Rev. 2017, 117, 4342– 4375,  DOI: 10.1021/acs.chemrev.6b00638
      24
      Manipulation and Motion of Organelles and Single Molecules in Living Cells
      Norregaard, Kamilla; Metzler, Ralf; Ritter, Christine M.; Berg-Soerensen, Kirstine; Oddershede, Lene B.
      Chemical Reviews (Washington, DC, United States) (2017), 117 (5), 4342-4375CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. The biomol. is among the most important building blocks of biol. systems, and a full understanding of its function forms the scaffold for describing the mechanisms of higher order structures as organelles and cells. Force is a fundamental regulatory mechanism of biomol. interactions driving many cellular processes. The forces on a mol. scale are exactly in the range that can be manipulated and probed with single mol. force spectroscopy. The natural environment of a biomol. is inside a living cell, hence, this is the most relevant environment for probing their function. In vivo studies are, however, challenged by the complexity of the cell. In this review, we start with presenting relevant theor. tools for analyzing single mol. data obtained in intracellular environments followed by a description of state-of-the art visualization techniques. The most commonly used force spectroscopy techniques, namely optical tweezers, magnetic tweezers, and at. force microscopy, are described in detail, and their strength and limitations related to in vivo expts. are discussed. Finally, recent exciting discoveries within the field of in vivo manipulation and dynamics of single mol. and organelles are reviewed.
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    25. 25
      Nishimura, Y.; Nishida, K.; Yamamoto, Y.; Ito, S.; Tokonami, S.; Iida, T. Control of submillimeter phase transition by collective photothermal effect. J. Phys. Chem. C 2014, 118, 18799– 18804,  DOI: 10.1021/jp506405w
      25
      Control of Submillimeter Phase Transition by Collective Photothermal Effect
      Nishimura, Yushi; Nishida, Keisuke; Yamamoto, Yojiro; Ito, Syoji; Tokonami, Shiho; Iida, Takuya
      Journal of Physical Chemistry C (2014), 118 (32), 18799-18804CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
      Local mol. states and biol. materials in small spaces ranging from the microscale to nanoscale can be modulated for medical and biol. applications using the photothermal effect (PTE). However, there have been only a few reports on exploiting the collective phenomena of localized surface plasmons (LSPs) to increase the amt. of light-induced heat for the control of material states and the generation of macroscopic assembled structures. Here, we clarify that microbeads covered with a vast no. of Ag nanoparticles can induce a large PTE and generate a submillimeter bubble within several tens of seconds under the synergetic effect of the light-induced force (LIF) and photothermal convection enhanced by collective phenomena of LSPs. Control of the phase transition induced by such a "collective photothermal effect" enables rapid assembling of macroscopic structures consisting of nanomaterials, which would be used for detection of a small amt. of proteins based on light-induced heat coagulation.
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    26. 26
      Iida, T. Development of Innovative Bio-measurement Technology by Micro-flow Light-Induced Acceleration; Horiba Tech Rep; 2021; Vol. 55, p 11.
      There is no corresponding record for this reference.
    27. 27
      Tokonami, S.; Iida, T. Review: Novel sensing strategies for bacterial detection based on active and passive methods driven by external field. Anal. Chim. Acta 2017, 988, 1– 16,  DOI: 10.1016/j.aca.2017.07.034
      There is no corresponding record for this reference.
    28. 28
      Tokonami, S.; Kurita, S.; Yoshikawa, R.; Sakurai, K.; Suehiro, T.; Yamamoto, Y.; Tamura, M.; Karthaus, O.; Iida, T. Light-induced assembly of living bacteria with honeycomb substrate. Sci. Adv. 2020, 6, eaaz5757  DOI: 10.1126/sciadv.aaz5757
      There is no corresponding record for this reference.
    29. 29
      Iida, T.; Hamatani, S.; Takagi, Y.; Fujiwara, K.; Tamura, M.; Tokonami, S. Attogram-level light-induced antigen-antibody binding confined in microflow. Commun. Biol. 2022, 5, 1053,  DOI: 10.1038/s42003-022-03946-0
      There is no corresponding record for this reference.
    30. 30
      Fujiwara, K.; Takagi, Y.; Tamura, M.; Omura, M.; Morimoto, K.; Nakase, I.; Tokonami, S.; Iida, T. Ultrafast sensitivity-controlled and specific detection of extracellular vesicles using optical force with antibody-modified microparticles in a microflow system. Nanoscale Horiz. 2023, 8, 1034,  DOI: 10.1039/D2NH00576J
      There is no corresponding record for this reference.
    31. 31
      Kanoda, M.; Hayashi, K.; Takagi, Y.; Tamura, M.; Tokonami, S.; Iida, T. High-throughput light-induced immunoassay with milliwatt-level laser under one-minute optical antibody-coating on nanoparticle-imprinted substrate. npj Biosensing 2024, 1, 1,  DOI: 10.1038/s44328-024-00004-z
      There is no corresponding record for this reference.
    32. 32
      Nakase, I.; Miyai, M.; Noguchi, K.; Tamura, M.; Yamamoto, Y.; Nishimura, Y.; Omura, M.; Hayashi, K.; Futaki, S.; Tokonami, S.; Iida, T. Light-Induced Condensation of Biofunctional Molecules around Targeted Living Cells to Accelerate Cytosolic Delivery. Nano Lett. 2022, 22, 9805– 9814,  DOI: 10.1021/acs.nanolett.2c02437
      32
      Light-Induced Condensation of Biofunctional Molecules around Targeted Living Cells to Accelerate Cytosolic Delivery
      Nakase, Ikuhiko; Miyai, Moe; Noguchi, Kosuke; Tamura, Mamoru; Yamamoto, Yasuyuki; Nishimura, Yushi; Omura, Mika; Hayashi, Kota; Futaki, Shiroh; Tokonami, Shiho; Iida, Takuya
      Nano Letters (2022), 22 (24), 9805-9814CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      The light-induced force and convection can be enhanced by the collective effect of electrons (superradiance and red shift) in high-d. metallic nanoparticles, leading to macroscopic assembly of target mols. We here demonstrate application of the light-induced assembly for drug delivery system with enhancement of cell membrane accumulation and penetration of biofunctional mols. including cell-penetrating peptides (CPPs) with superradiance-mediated photothermal convection. For induction of photothermal assembly around targeted living cells in cell culture medium, IR continuous-wave laser light was focused onto high-d. gold-particle-bound glass bottom dishes exhibiting plasmonic superradiance or thin gold-film-coated glass bottom dishes. In this system, the biofunctional mols. can be concd. around the targeted living cells and internalized into them only by 100 s laser irradn. Using this simple approach, we successfully achieved enhanced cytosolic release of the CPPs and apoptosis induction using a pro-apoptotic domain with a very low peptide concn. (nM level) by light-induced condensation.
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    33. 33
      Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature 1996, 382, 607– 609,  DOI: 10.1038/382607a0
      33
      A DNA-based method for rationally assembling nanoparticles into macroscopic materials
      Mirkin, Chad A.; Letsinger, Robert L.; Mucic, Robert C.; Storhoff, James J.
      Nature (London) (1996), 382 (6592), 607-609CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)
      Colloidal particles of metals and semiconductors have potentially useful optical, optoelectronic and material properties that derive from their small (nanoscopic) size. These properties might lead to applications including chem. sensors, spectroscopic enhancers, quantum dot and nanostructure fabrication, and microimaging methods. A great deal of control can now be exercised over the chem. compn., size and polydispersity of colloidal particles, and many methods have been developed for assembling them into useful aggregates and materials. Here we describe a method for assembling colloidal gold nanoparticles rationally and reversibly into macroscopic aggregates. The method involves attaching to the surfaces of two batches of 13-nm gold particles non-complementary DNA oligonucleotides capped with thiol groups, which bind to gold. When we add to the soln. an oligonucleotide duplex with 'sticky ends' that are complementary to the two grafted sequences, the nanoparticles self-assemble into aggregates. This assembly process can be reversed by thermal denaturation. This strategy should now make it possible to tailor the optical, electronic and structural properties of the colloidal aggregates by using the specificity of DNA interactions to direct the interactions between particles of different size and compn.
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    34. 34
      Alivisatos, A. P.; Johnsson, K. P.; Peng, X.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P.; Schultz, P. G. Organization of ’nanocrystal molecules’ using DNA. Nature 1996, 382, 609– 611,  DOI: 10.1038/382609a0
      34
      Organization of 'nanocrystal molecules' using DNA
      Alivisatos, A. Paul; Johnsson, Kai P.; Peng, Xiaogang; Wilson, Troy E.; Loweth, Colin J.; Bruchez, Marcel P., Jr.; Schultz, Peter G.
      Nature (London) (1996), 382 (6592), 609-611CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)
      The authors describe a strategy for the synthesis of 'nanocrystal mols.', in which discrete nos. of Au nanocrystals are organized into spatially defined structures based on Watson-Crick base-pairing interactions. The authors attach single-stranded DNA oligonucleotides of defined length and sequence to individual nanocrystals, and these assemble into dimers and trimers on addn. of a complementary single-stranded DNA template. The authors anticipate that this approach should allow the construction of more complex two- and three-dimensional assemblies.
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    35. 35
      Pinheiro, A.; Han, D.; Shih, W.; Yan, H. Challenges and opportunities for structural DNA nanotechnology. Nat. Nanotechnol. 2011, 6, 763– 772,  DOI: 10.1038/nnano.2011.187
      35
      Challenges and opportunities for structural DNA nanotechnology
      Pinheiro, Andre V.; Han, Dongran; Shih, William M.; Yan, Hao
      Nature Nanotechnology (2011), 6 (12), 763-772CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      A review. DNA mols. have been used to build a variety of nanoscale structures and devices over the past 30 years, and potential applications have begun to emerge. But the development of more advanced structures and applications will require a no. of issues to be addressed, the most significant of which are the high cost of DNA and the high error rate of self-assembly. Here we examine the tech. challenges in the field of structural DNA nanotechnol. and outline some of the promising applications that could be developed if these hurdles can be overcome. In particular, we highlight the potential use of DNA nanostructures in mol. and cellular biophysics, as biomimetic systems, in energy transfer and photonics, and in diagnostics and therapeutics for human health.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsVagu7%252FL&md5=f23efb8658948ab55fdd3a32ce42686b
    36. 36
      Mu, B.; Zhang, J.; McNicholas, T. P.; Reuel, N. F.; Kruss, S.; Strano, M. S. Recent advances in molecular recognition based on nanoengineered platforms. Acc. Chem. Res. 2014, 47, 979– 988,  DOI: 10.1021/ar400162w
      36
      Recent Advances in Molecular Recognition Based on Nanoengineered Platforms
      Mu, Bin; Zhang, Jingqing; McNicholas, Thomas P.; Reuel, Nigel F.; Kruss, Sebastian; Strano, Michael S.
      Accounts of Chemical Research (2014), 47 (4), 979-988CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review. Nanoparticles and nanoengineered platforms have great potential for technologies involving biomoleuclar detection or cell-related biosensing, and have provided effective chem. interfaces for mol. recognition. Typically, chemists work on the modification of synthetic polymers or macromols., which they link to the nanoparticles by covalent or noncovalent approaches. The motivation for chem. modification is to enhance the selectivity and sensitivity, and to improve the biocompatibility for the in vivo applications. In this Account, we present recent advances in the development and application of chem. interfaces for mol. recognition for nanoparticles and nanoengineered platforms, in particular single-walled carbon nanotubes (SWNTs). We discuss emerging approaches for recognizing small mols., glycosylated proteins, and serum biomarkers. For example, we compare and discuss detection methods for ATP, NO, H2O2, and monosaccharides for recent nanomaterials. Fluorometric detection appears to have great potential for quantifying concn. gradients and detg. their location in living cells. For macromol. detection, new methods for glycoprofiling using such interfaces appear promising, and benefit specifically from the potential elimination of cumbersome labeling and liberation steps during conventional anal. of glycans, augmenting the currently used mass spectrometry (MS), capillary electrophoresis (CE), and liq. chromatog. (LC) methods. In particular, we demonstrated the great potential of fluorescent SWNTs for glycan-lectin interactions sensing. In this case, SWNTs are noncovalently functionalized to introduce a chelated nickel group. This group provides a docking site for the His-tagged lectin and acts as the signal modulator. As the nickel proximity to the SWNT surface changes, the fluorescent signal is increased or attenuated. When a free glycan or glycosylated probe interacts with the lectin, the signal increases and they are able to obtain loading curves similar to surface plasmon resonance measurements. They demonstrate the sensitivity and specificity of this platform with two higher-affined glycan-lectin pairs: fucose (Fuc) to PA-IIL and N-acetylglucosamine (GlcNAc) to GafD. Lastly, we discuss how developments in protein biomarker detection in general are benefiting specifically from label-free mol. recognition. Elec. field effect transistors, chemi-resistive and fluorometric nanosensors based on various nanomaterials have demonstrated substantial progress in recent years in addressing this challenging problem. In this Account, we compare the balance between sensitivity, selectivity, and nonspecific adsorption for various applications. In particular, our group has utilized SWNTs as fluorescence sensors for label-free protein-protein interaction measurements. In this assay, we have encapsulated each nanotube in a biocompatible polymer, chitosan, which has been further modified to conjugate nitrilotriacetic acid (NTA) groups. After Ni2+ chelation, NTA Ni2+ complexes bind to his-tagged proteins, resulting in a local environment change of the SWNT array, leading to optical fluorescence modulation with detection limit down to 100 nM. We have further engineered the platform to monitor single protein binding events, with an even lower detection limit down to 10 pM.
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    37. 37
      Seaberg, J.; Montazerian, H.; Hossen, M. N.; Bhattacharya, R.; Khademhosseini, A.; Mukherjee, P. Hybrid nanosystems for biomedical applications. ACS Nano 2021, 15 (2), 2099– 2142,  DOI: 10.1021/acsnano.0c09382
      37
      Hybrid Nanosystems for Biomedical Applications
      Seaberg, Joshua; Montazerian, Hossein; Hossen, Md Nazir; Bhattacharya, Resham; Khademhosseini, Ali; Mukherjee, Priyabrata
      ACS Nano (2021), 15 (2), 2099-2142CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      A review. Inorg./org. hybrid nanosystems have been increasingly developed for their versatility and efficacy at overcoming obstacles not readily surmounted by nonhybridized counterparts. Currently, hybrid nanosystems are implemented for gene therapy, drug delivery, and phototherapy in addn. to tissue regeneration, vaccines, antibacterials, biomol. detection, imaging probes, and theranostics. Though diverse, these nanosystems can be classified according to foundational inorg./org. components, accessory moieties, and architecture of hybridization. Within this Review, we begin by providing a historical context for the development of biomedical hybrid nanosystems before describing the properties, synthesis, and characterization of their component building blocks. Afterward, we introduce the architectures of hybridization and highlight recent biomedical nanosystem developments by area of application, emphasizing hybrids of distinctive utility and innovation. Finally, we draw attention to ongoing clin. trials before recapping our discussion of hybrid nanosystems and providing a perspective on the future of the field.
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    38. 38
      Tokonami, S.; Shiigi, H.; Nagaoka, T. Open bridge-structured gold nanoparticle array for label-free DNA detection. Anal. Chem. 2008, 80, 8071– 8075,  DOI: 10.1021/ac801088u
      38
      Open Bridge-Structured Gold Nanoparticle Array for Label-Free DNA Detection
      Tokonami, Shiho; Shiigi, Hiroshi; Nagaoka, Tsutomu
      Analytical Chemistry (Washington, DC, United States) (2008), 80 (21), 8071-8075CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      We focused on changes in the elec. property of the open bridge-structured gold nanoparticles array consisting of 46-nm parent and 12-nm son gold nanoparticles by hybridization and applied it for a simple elec. DNA detection. Since a target DNA of a 24-mer oligonucleotide was added to the probe DNA modified 12-nm Au nanoparticles, which was arranged on the gap between the 46-nm Au particles, the response was read by an elec. readout system. Even in a simple measuring method, we obtained a rapid response to the cDNA with a high S/N ratio of 30 over a wide concn. range and a detection limit of 5.0 fmol. Moreover, the array discriminated 1-base mismatches, regardless of their location in the DNA sequence, which enabled us to detect single-nucleotide polymorphism, which is one of the important diagnoses, without any polymerase chain reaction amplification, sophisticated instrumentation, or fluorescent labeling through an easy-to-handle elec. readout system.
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    39. 39
      Iida, T.; Nishimura, Y.; Tamura, M.; Nishida, K.; Ito, S.; Tokonami, S. Submillimetre Network Formation by Light-induced Hybridization of Zeptomole-level DNA. Sci. Rep 2016, 6, 37768,  DOI: 10.1038/srep37768
      39
      Submillimetre Network Formation by Light-induced Hybridization of Zeptomole-level DNA
      Iida, Takuya; Nishimura, Yushi; Tamura, Mamoru; Nishida, Keisuke; Ito, Syoji; Tokonami, Shiho
      Scientific Reports (2016), 6 (), 37768CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      Macroscopic unique self-assembled structures are produced via double-stranded DNA formation (hybridization) as a specific binding essential in biol. systems. However, a large amt. of complementary DNA mols. are usually required to form an optically observable structure via natural hybridization, and the detection of small amts. of DNA less than femtomole requires complex and time-consuming procedures. Here, we demonstrate the laser-induced acceleration of hybridization between zeptomole-level DNA and DNA-modified nanoparticles (NPs), resulting in the assembly of a submillimetre network-like structure at the desired position with a dramatic spectral modulation within several minutes. The gradual enhancement of light-induced force and convection facilitated the two-dimensional network growth near the air-liq. interface with optical and fluidic symmetry breakdown. The simultaneous microscope observation and local spectroscopy revealed that the assembling process and spectral change are sensitive to the DNA sequence. Our findings establish innovative guiding principles for facile bottom-up prodn. via various biomol. recognition events.
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    40. 40
      Iida, T.; Ishihara, H. Theory of resonant radiation force exerted on nanostructures by optical excitation of their quantum states: From microscopic to macroscopic descriptions. Phys. Rev. B 2008, 77, 245319,  DOI: 10.1103/PhysRevB.77.245319
      40
      Theory of resonant radiation force exerted on nanostructures by optical excitation of their quantum states: From microscopic to macroscopic descriptions
      Iida, Takuya; Ishihara, Hajime
      Physical Review B: Condensed Matter and Materials Physics (2008), 77 (24), 245319/1-245319/16CODEN: PRBMDO; ISSN:1098-0121. (American Physical Society)
      The authors establish a theor. framework that provides a bridge between the microscopic to macroscopic descriptions of the radiation force (RF) under a quantum resonance condition. By using this framework, an explicit anal. expression is derived to clearly demonstrate the properties of the resonant RF on nanostructures and related novel phenomena. For a single nano-object, the RF drastically changes with the size, shape, and quality of a nano-object due to the spatial correlations of the internal radiation field and the matter-excited states. This property is highly advantageous in the selective manipulation of quantum properties of nano-objects. For multiple nano-objects, an attractive (repulsive) inter-object radiation force (IRF) arises between nano-objects under the optical excitation of a particular coupled state of their spatially sepd. polaritons, and this state is termed as "polaritonic mol.". This IRF can be enhanced even between the nano-objects that have a large spatial sepn. if there exist intermediate nano-objects even with very weak induced polarizations, and this effect is termed as "superinterobject radiation force.". In addn., a neg. dissipative force arises when the electronic polarization in a particular nano-object is inverted by a photo-mediated interaction. Since the resonant RF and IRF depend on many degrees of freedom of both nanostructures and light, they will provide a great variety of optical control methods for the collective dynamics of nanocomposite materials.
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    41. 41
      Hosokawa, C.; Tsuji, T.; Kishimoto, T.; Okubo, T.; Kudoh, S. N.; Kawano, S. Convection dynamics forced by optical trapping with a focused laser beam. J. Phys. Chem. C 2020, 124, 8323– 8333,  DOI: 10.1021/acs.jpcc.9b11663
      41
      Convection Dynamics Forced by Optical Trapping with a Focused Laser Beam
      Hosokawa, Chie; Tsuji, Tetsuro; Kishimoto, Tatsunori; Okubo, Takumi; Kudoh, Suguru N.; Kawano, Satoyuki
      Journal of Physical Chemistry C (2020), 124 (15), 8323-8333CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
      Optical trapping dynamics of colloidal particles in soln. is essential for understanding laser-induced assembling of mols. and nanomaterials, which contributes to nanofabrication, bioengineering, and microfluidics. The importance of the surrounding fluid motion in optical trapping is studied; i.e., convection fluid dynamics forced by optical trapping with a focused laser beam is revealed. The fluid flow in optical trapping is evaluated by both expts. using the particle-image-velocimetry of fluorescent particles in solns. and theor. consideration based on numerical anal. A theor. model consists of Navier-Stokes equations with the Boussinesq approxn. that considers the temp. elevation induced by a photothermal effect. The effect of the particle motion induced by the optical force on fluid flow is also included in the anal. by developing a simple 1-way homogeneous-type multiphase flow model. From both exptl. and theor. results, the fluid flow in optical trapping is caused not only by thermal convection due to the temp. elevation but also by the collective particle motion induced by optical forces. The optical forces can induce the large-scale fluid convection, which supports accumulating the target particles to the focal spot.
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    42. 42
      Baffou, G.; Quidant, R. Thermo-plasmonics: Using metallic nanostructures as nano-sources of heat. Laser Photonics Rev. 2013, 7, 171– 187,  DOI: 10.1002/lpor.201200003
      42
      Thermo-plasmonics: using metallic nanostructures as nano-sources of heat
      Baffou, Guillaume; Quidant, Romain
      Laser & Photonics Reviews (2013), 7 (2), 171-187CODEN: LPRAB8; ISSN:1863-8880. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Recent years have seen a growing interest in using metal nanostructures to control temp. on the nanoscale. Under illumination at its plasmonic resonance, a metal nanoparticle features enhanced light absorption, turning it into an ideal nano-source of heat, remotely controllable using light. Such a powerful and flexible photothermal scheme is the basis of thermo-plasmonics. Here, the recent progress of this emerging and fast-growing field is reviewed. First, the physics of heat generation in metal nanoparticles is described, under both continuous and pulsed illumination. The second part is dedicated to numerical and exptl. methods that have been developed to further understand and engineer plasmonic-assisted heating processes on the nanoscale. Finally, some of the most recent applications based on the heat generated by gold nanoparticles are surveyed, namely photothermal cancer therapy, nano-surgery, drug delivery, photothermal imaging, protein tracking, photoacoustic imaging, nano-chem. and optofluidics.
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    43. 43
      Hastman, D. A.; Melinger, J. S.; Aragonés, G. L.; Cunningham, P. D.; Chiriboga, M.; Salvato, Z. J.; Salvato, T. M.; Brown, C. W.; Mathur, D.; Medintz, I. L.; Oh, E.; Diaz, S. A. Femtosecond laser pulse excitation of DNA-labeled gold nanoparticles: establishing a quantitative local nanothermometer for biological applications. ACS Nano 2020, 14 (7), 8570– 8583,  DOI: 10.1021/acsnano.0c02899
      43
      Femtosecond Laser Pulse Excitation of DNA-Labeled Gold Nanoparticles: Establishing a Quantitative Local Nanothermometer for Biological Applications
      Hastman, David A.; Melinger, Joseph S.; Aragones, Guillermo Lasarte; Cunningham, Paul D.; Chiriboga, Matthew; Salvato, Zachary J.; Salvato, Thomas M.; Brown, Carl W.; Mathur, Divita; Medintz, Igor L.; Oh, Eunkeu; Diaz, Sebastian A.
      ACS Nano (2020), 14 (7), 8570-8583CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      Femtosecond (fs) laser pulsed excitation of plasmonic nanoparticle (NP)-biomol. conjugates is a promising method to locally heat biol. materials. Studies have demonstrated that fs pulses of light can modulate the activity of DNA or proteins when attached to plasmonic NPs; however, the precision over subsequent biol. function remains largely undetd. Specifically, the temp. the localized biomols. "experience" remains unknown. The authors used 55 nm gold nanoparticles (AuNPs) displaying double-stranded (ds) DNA to examine how, for dsDNA with different melting temps., the laser pulse energy fluence and bulk soln. temp. affect the rate of local DNA denaturation. A universal "template" single-stranded DNA was attached to the AuNP surface, and three dye-labeled probe strands, distinct in length and melting temp., were hybridized to it creating three individual dsDNA-AuNP bioconjugates. The dye-labeled probe strands were used to quantify the rate and amt. of DNA release after a given no. of light pulses, which was then correlated to the dsDNA denaturation temp., resulting in a quant. nanothermometer. The localized DNA denaturation rate could be modulated by more than threefold over the biol. relevant range of 8-53° by varying pulse energy fluence, DNA melting temp., and surrounding bath temp. With a modified dissocn. equation tailored for this system, a "sensed" temp. parameter was extd. and compared to simulated AuNP temp. profiles. Detg. actual biol. responses in such systems can allow researchers to design precision nanoscale photothermal heating sources.
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    44. 44
      Wen, S.; Miao, X.; Fan, G.-C.; Xu, T.; Jiang, L.-P.; Wu, P.; Cai, C.; Zhu, J.-J. Aptamer-Conjugated Au Nanocage/SiO2 Core-Shell Bifunctional Nanoprobes with High Stability and Biocompatibility for Cellular SERS Imaging and Near-Infrared Photothermal Therapy. ACS Sens. 2019, 4 (2), 301– 308,  DOI: 10.1021/acssensors.8b00682
      44
      Aptamer-Conjugated Au Nanocage/SiO2 Core-Shell Bifunctional Nanoprobes with High Stability and Biocompatibility for Cellular SERS Imaging and Near-Infrared Photothermal Therapy
      Wen Shengping; Miao Xuran; Xu Tingting; Jiang Li-Ping; Zhu Jun-Jie; Fan Gao-Chao; Wu Ping; Cai Chenxin
      ACS sensors (2019), 4 (2), 301-308 ISSN:.
      The combination of surface-enhanced Raman scattering (SERS) imaging technology with near-infrared (NIR) light-triggered photothermal therapy is of utmost importance to develop novel theranostic platforms. Herein, an aptamer-conjugated Au nanocage/SiO2 (AuNC/SiO2/Apt) core-shell Raman nanoprobe has been rationally designed as the bifunctional theranostic platform to fulfill this task. In this theranostic system, the Raman-labeled Au nanocage (AuNC) was encapsulated into a bioinert shell of SiO2, followed by conjugating aptamer AS1411 as the target-recognition moiety. AuNC served as the SERS-active and photothermal substrate due to its large free volume, built-in plasmon effect, and NIR photothermal capacity, while the SiO2 coating endowed the nanoprobes with good stability and biocompatibility, as well as abundant anchoring sites for surface functionalization. Considering their prominent SERS and photothermal properties, the application potential of the AuNC/SiO2/Apt nanoprobes was investigated. The proposed nanoprobes could be applied to targeted detection and SERS imaging of nucleolin-overexpressing cancer cells (MCF-7 cells as the model) from normal cells and also exhibited acceptable photothermal efficacy without systematic toxicity. This theranostic nanoplatform provided a possible opportunity for in situ diagnosis and noninvasive treatment of cancer cells by SERS imaging-guided photothermal therapy.
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    45. 45
      Kojima, C.; Watanabe, Y.; Hattori, H.; Iida, T. Design of Photosensitive Gold Nanoparticles for Biomedical Applications Based on Self-Consistent Optical Response Theory. J. Phys. Chem. C 2011, 115, 19091– 19095,  DOI: 10.1021/jp206501h
      45
      Design of Photosensitive Gold Nanoparticles for Biomedical Applications Based on Self-Consistent Optical Response Theory
      Kojima, Chie; Watanabe, Yasutaka; Hattori, Hironori; Iida, Takuya
      Journal of Physical Chemistry C (2011), 115 (39), 19091-19095CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
      Gold nanoparticles (Au NPs) have been studied for both photothermal therapy and imaging. Efficient photothermogenic response to near-IR light is necessary for in vivo applications. The photosensitive properties of Au NPs were theor. analyzed by self-consistent treatment of Maxwell's equation. With the Au concn. held const., a single Au NP with a diam. of 60 nm had the most efficient photothermogenic properties by 532 nm laser irradn. Particularly, due to the multiple interactions of mirror images of localized surface plasmons, closely spaced multiple Au NPs exhibited enhanced photothermogenic properties in the longer wavelength region even when the Au NPs were small. A comparison of the theor. and exptl. results suggests that the multiple Au NPs are created by the seeding growth of Au NPs in the PEGylated dendrimer. These results provide the guiding principles for design of Au NPs suitable for photorelated biomedical applications.
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    46. 46
      Iida, T. Control of Plasmonic Superradiance in Metallic Nanoparticle Assembly by Light-Induced Force and Fluctuations. J. Phys. Chem. Lett. 2012, 3, 332– 336,  DOI: 10.1021/jz2014924
      46
      Control of Plasmonic Superradiance in Metallic Nanoparticle Assembly by Light-Induced Force and Fluctuations
      Iida, Takuya
      Journal of Physical Chemistry Letters (2012), 3 (3), 332-336CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
      The possibility of simultaneous control of the configuration and optical functions of a metallic nanoparticle (NP) assembly by light-induced force (LIF) and thermal fluctuations has been demonstrated on the basis of self-consistent theory of LIF and nonequil. dynamics. It has been clarified that the NPs are arranged parallel to the polarization of the focused laser beam under the balance of LIF and the electrostatic repulsive force due to the ions on the surface of NPs. Particularly, in such a NP assembly consisting of high-d. NPs, the light-scattering rate (radiative decay) of localized surface plasmon polaritons (LSPPs) can be drastically enhanced to be greater than 100 meV (10 times that of single NPs), and the spectral width is also greatly broadened due to the superradiance effect. The results will provide a foundation of the principles for designing a NP assembly with controllable light scattering for highly efficient broad-band light energy conversion devices.
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    47. 47
      Tokonami, S.; Hidaka, S.; Nishida, K.; Yamamoto, Y.; Nakao, H.; Iida, T. Multipole Superradiance from Densely Assembled Metallic Nanoparticles. J. Phys. Chem. C 2013, 117, 15247– 15252,  DOI: 10.1021/jp4028244
      47
      Multipole Superradiance from Densely Assembled Metallic Nanoparticles
      Tokonami, Shiho; Hidaka, Shimpei; Nishida, Keisuke; Yamamoto, Yojiro; Nakao, Hidenobu; Iida, Takuya
      Journal of Physical Chemistry C (2013), 117 (29), 15247-15252CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
      The collective phenomenon of localized surface plasmons (LSPs) in a high-d. collection of interacting metallic nanoparticles (NPs) is a crucial issue in various research fields such as optical physics, photochem., and biol. science. Here, we report the dark-field measurement of the chem. controlled optical response of LSPs in densely assembled collection of a vast no. of gold NPs on a microsphere (AuNP-covered bead). Remarkably, AuNP-covered beads exhibit plasmonic superradiance depending on sizes of binder mols., where the giant spectral broadening more than 400 meV and significant enhancement of scattering have been obsd. Furthermore, self-consistent theor. anal. has also revealed that multipole collective modes contribute to the superradiance, leading to the enhancement by 2 orders of magnitude in both the far-field scattering and the localized fields of broadband light. The results obtained provide an innovative design principle for solar energy conversion and optical biosensors with incoherent light.
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    48. 48
      Sunaga, N.; Miura, Y.; Kasahara, N.; Sakurai, R. Targeting oncogenic KRAS in non-small-cell lung cancer. Cancers 2021, 13, 5956,  DOI: 10.3390/cancers13235956
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssensors.4c02119.

    • Time course of transmission images with the heterogeneous probe-particles and target DNA without laser irradiation, fluorescence intensity for three experimental conditions, optical transmission image of the photothermal effect, theoretically calculated optical absorption of gold nanoparticles, stability, and functionality of probes, confirmation of the photobleaching effect of target DNA, experimentally observed absorption spectrum of AuNPs, surface density of probe DNA, schematic illustrations of experimental setup, DNA sequences, and explanation of the movie (PDF)

    • Movie of the optical transmission during optical condensation (MP4)


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