Melanoma Detection by AFM Indentation of Histological Specimens.

Melanoma is visible unlike other types of cancer, but it is still challenging to diagnose correctly because of the difficulty in distinguishing between benign nevus and melanoma. We conducted a robust investigation of melanoma, identifying considerable differences in local elastic properties between nevus and melanoma tissues by using atomic force microscopy (AFM) indentation of histological specimens. Specifically, the histograms of the elastic modulus of melanoma displayed multimodal Gaussian distributions, exhibiting heterogeneous mechanical properties, in contrast with the unimodal distributions of elastic modulus in the benign nevus. We identified this notable signature was consistent regardless of blotch incidence by sex, age, anatomical site (e.g., thigh, calf, arm, eyelid, and cheek), or cancer stage (I, IV, and V). In addition, we found that the non-linearity of the force-distance curves for melanoma is increased compared to benign nevus. We believe that AFM indentation of histological specimens may technically complement conventional histopathological analysis for earlier and more precise melanoma detection.

Conformation Control of Amyloid Filaments by Repeated Thermal Perturbation.

We report that repeated thermal perturbation by thermal cycling (TC) accelerates the formation rate of amyloid filaments at microliter volumes (10-200 µL) and produces a new conformation of zigzag-shaped filaments. The amyloid filaments have been synthesized under different TC conditions, such as temperature variations (ΔT = 0-86 °C) and the number of cycles (C# = 30-90). In particular, the filament formation was promoted by TC with ΔT ≥ 30 °C. This indicates that the change in binding energy of ß-sheets and the breakage of disulfide bonds induced by TC with large ΔT contributed to the increased filament growth. This molecular interaction was investigated by molecular dynamics simulation. We also found that TC leads to the formation of amyloid filaments with peculiar conformation (zigzag-shaped filaments). Moreover, key structural parameters (tortuosity, segment length, and joint angle) of the amyloid filaments could be fine-tuned by selecting certain ΔT conditions. Taken together, we confirmed that the TC not only promotes the formation of amyloid filaments but also affects the conformational changes of the filaments.

Surface potential microscopy of surfactant-controlled single gold nanoparticle.

The surface potential of nanoparticles plays a key role in numerous applications, such as drug delivery and cellular uptake. The estimation of the surface potential of nanoparticles as drug carriers or contrast agents is important for the design of nanoparticle-based biomedical platforms. Herein, we report the direct measurement of the surface potential of individual gold nanorods (GNRs) via Kelvin probe force microscopy (KPFM) at the nanoscale. GNRs were capped by a surfactant, cetyltrimethylammonium bromide (CTAB), which was removed by centrifugation. CTAB removal is essential for GNR-based biomedical applications because of the cytotoxicity of CTAB. Applying KPFM analysis, we found that the mean surface potential of the GNRs became more negative as the CTAB was removed from the GNR. The results indicate that the negative charge of GNRs is covered by the electrostatic charge of the CTAB molecules. Similar trends were observed in experiments with gold nanospheres (GNS) capped by citrates. Overall, KPFM-based techniques characterize the surfactant of individual nanoparticles (i.e. GNR or GNS) with high resolution by mapping the surface potential of a single nanoparticle, which aids in designing engineered nanoparticles for biomedical applications.

MoS2 Field-Effect Transistor-Amyloid-ß1-42 Hybrid Device for Signal Amplified Detection of MMP-9.

The detection of circulating protein (CP) is very important for the diagnosis and therapeutics of cancer. Conventional techniques based on a specific antibody-antigen interaction are still lacking because of a shortage of cost effectiveness, complicated sandwich structure and tagging process, and inconsistent detection of CP due to the inherent instability of antibodies. Herein, we demonstrate a hybrid device consisting of two-dimensional (2D) nanoscale molybdenum disulfide (MoS2) field-effect transistor (FET) with an amyloid-ß1-42 (Aß1-42) functionalized surface, which amplifies electric signals of the FET in order to detect matrix metalloproteinase-9 (MMP-9), which is a certain type of CP that degrades Aß1-42. With the hybrid device, we detected the concentrations of MMP-9 in the range from 1 pM to 10 nM. Moreover, using tapping-mode atomic force microscopy and Kelvin probe force microscopy, we verified that the signal amplification corresponding to the MMP-9 concentrations was caused by the reduced length and the decreased surface potential of degraded Aß1-42 due to MMP-9. The hybrid device studied in this paper can be very useful for monitoring MMP-9 activity, as well as serving as a sensing platform for the electrical signal amplification of 2D MoS2 FET-biosensors.

Extremely sensitive and wide-range silver ion detection via assessing the integrated surface potential of a DNA-capped gold nanoparticle.

With the rapid development of nanotechnology and its associated waste stream, public concern is growing over the potential toxicity exposure to heavy metal ions poses to the human body and the environment. Herein, we report an extremely sensitive Kelvin probe force microscopy (KPFM)-based platform for detecting nanotoxic materials (e.g. Ag+) accomplished by probing the integrated surface potential differences of a single gold nanoparticle on which an interaction between probe DNA and target DNA occurs. This interaction can amplify the surface potential of the nanoparticle owing to the coordination bond mediated by Ag+ (cytosine-Ag+-cytosine base pairs). Interestingly, compared with conventional methods, this platform is capable of extremely sensitive Ag+ detection (∼1 fM) in a remarkably wide-range (1 fM to 1 µM). Furthermore, this platform enables Ag+ detection in a practical sample (general drinking water), and this KPFM-based technique may have the potential to detect other toxic heavy metal ions and single nucleotide polymorphisms by designing specific DNA sequences.

Identifying DNA mismatches at single-nucleotide resolution by probing individual surface potentials of DNA-capped nanoparticles.

Here, we demonstrate a powerful method to discriminate DNA mismatches at single-nucleotide resolution from 0 to 5 mismatches (χ0 to χ5) using Kelvin probe force microscopy (KPFM). Using our previously developed method, we quantified the surface potentials (SPs) of individual DNA-capped nanoparticles (DCNPs, ∼100 nm). On each DCNP, DNA hybridization occurs between ∼2200 immobilized probe DNA (pDNA) and target DNA with mismatches (tDNA, ∼80 nM). Thus, each DCNP used in the bioassay (each pDNA-tDNA interaction) corresponds to a single ensemble in which a large number of pDNA-tDNA interactions take place. Moreover, one KPFM image can scan at least dozens of ensembles, which allows statistical analysis (i.e., an ensemble average) of many bioassay cases (ensembles) under the same conditions. We found that as the χn increased from χ0 to χ5 in the tDNA, the average SP of dozens of ensembles (DCNPs) was attenuated owing to fewer hybridization events between the pDNA and the tDNA. Remarkably, the SP attenuation vs. the χn showed an inverse-linear correlation, albeit the equilibrium constant for DNA hybridization exponentially decreased asymptotically as the χn increased. In addition, we observed a cascade reaction at a 100-fold lower concentration of tDNA (∼0.8 nM); the average SP of DCNPs exhibited no significant decrease but rather split into two separate states (no-hybridization vs. full-hybridization). Compared to complementary tDNA (i.e., χ0), the ratio of no-hybridization/full-hybridization within a given set of DCNPs became ∼1.6 times higher in the presence of tDNA with single mismatches (i.e., χ1). The results imply that our method opens new avenues not only in the research on the DNA hybridization mechanism in the presence of DNA mismatches but also in the development of a robust technology for DNA mismatch detection.

Label-Free and Recalibrated Multilayer MoS2 Biosensor for Point-of-Care Diagnostics.

Molybdenum disulfide (MoS2) field-effect transistor (FET)-based biosensors have attracted significant attention as promising candidates for highly sensitive, label-free biomolecule detection devices. In this paper, toward practical applications of biosensors, we demonstrate reliable and quantitative detection of a prostate cancer biomarker using the MoS2 FET biosensor in a nonaqueous environment by reducing nonspecific molecular binding events and realizing uniform chemisorption of anti-PSA onto the MoS2 surface. A systematic and statistical study on the capability of the proposed device is presented, and the biological binding events are directly confirmed and characterized through intensive structural and electrical analysis. Our proposed biosensor can reliably detect various PSA concentrations with a limit of 100 fg/mL. Moreover, rigorous theoretical simulations provide a comprehensive understanding of the operating mechanism of the MoS2 FET biosensors, and further suggests the enhancement of the sensitivity through engineering device design parameters.

Blood Droplet-Based Cancer Diagnosis via Proteolytic Activity Measurement in Cancer Progression.

Matrix metalloproteinase (MMP) is a key marker and target molecule for cancer diagnosis, as MMP is able to cleave peptide chains resulting in degradation of extracellular matrix (ECM), a necessary step for cancer development. In particular, MMP2 has recently been recognized as an important biomarker for lung cancer. Despite the important role of detecting MMP molecules in cancer diagnosis, it is a daunting task to quantitatively understand a correlation between the status of cancer development and the secretion level of MMP in a blood droplet. Here, we demonstrate a nanoscale cancer diagnosis by nanomechanical quantitation of MMP2 molecules under cancer progression with using a blood droplet of lung cancer patients. Specifically, we measured the frequency dynamics of nanomechanical biosensor functionalized with peptide chains mimicking ECM in response to MMP2 secreted from tumors in lung with different metastasis level. It is shown that the frequency shift of the biosensor, which exhibits the detection sensitivity below 1 nM, enables the quantitation of the secretion level of MMP2 molecules during the progression of cancer cells or tumor growth. More importantly, using a blood droplet of lung cancer patients, nanomechanical biosensor is shown to be capable of depicting the correlation between the secretion level of MMP2 molecules and the level of cancer metastasis, which highlights the cantilever-based MMP2 detection for diagnosis of lung cancer. Our finding will broaden the understanding of cancer development activated by MMP and allow for a fast and point-of-care cancer diagnostics.

Dielectrophoretic force measurement of red blood cells exposed to oxidative stress using optical tweezers and a microfluidic chip.

Red blood cell (RBC) dysfunction is often associated with a pathological intervention, and it has been proposed as a critical risk factor for certain lethal diseases. Examining the cell viability of RBCs under various physiological conditions is essential and of importance for precise diagnosis and drug discovery in the field of medicine and pharmacy. In this paper, we report a new analytical method that employs dielectrophoretic (DEP) force measurements in absolute units to assess the viability, and potentially the functionality of RBCs. We precisely quantify the frequency-dependent DEP forces of the RBCs by using a micro-electrode embedded chip combined with optical tweezers. DEP characteristics are known to be well-correlated with the viability of biological cells, and DEP forces are measured in both fresh and long-term stored RBCs to investigate the effect that the storage period has on the cell viability. Moreover, we investigate the DEP behavior of RBCs when exposed to oxidative stress and verify whether EDTA protects the RBCs from an oxidant. From the experiments, it is found that the fresh RBCs without oxidative stress display very high DEP forces over the entire frequency range, exhibiting two cutoff frequencies. However, both the RBCs stored for the long-term period and exposed to oxidative stress reveals that there exist no significant DEP forces over the frequency range. The results indicate that the DEP forces can serve as a useful parameter to verify whether the RBCs in certain blood are fresh and not exposed to oxidative stress. Therefore, it is believed that our system can be applied to a diagnostic system to monitor the cell viability of the RBCs or other types of cells.

Detection of Silver Ions Using Dielectrophoretic Tweezers-Based Force Spectroscopy.

Understanding of the interactions of silver ions (Ag+) with polynucleotides is important not only to detect Ag+ over a wide range of concentrations in a simple, robust, and high-throughput manner but also to investigate the intermolecular interactions of hydrogen and coordinate interactions that are generated due to the interplay of Ag+, hydrogen ions (H+), and polynucleotides since it is critical to prevent adverse environmental effects that may cause DNA damage and develop strategies to treat this damage. Here, we demonstrate a novel approach to simultaneously detect Ag+ satisfying the above requirements and examine the combined intermolecular interactions of Ag+-polycytosine and H+-polycytosine DNA complexes using dielectrophoretic tweezers-based force spectroscopy. For this investigation, we detected Ag+ over a range of concentrations (1 nM to 100 µM) by quantifying the rupture force of the combined interactions and examined the interplay between the three factors (Ag+, H+, and polycytosine) using the same assay for the detection of Ag+. Our study provides a new avenue not only for the detection of heavy metal ions but also for the investigation of heavy metal ions-polynucleotide DNA complexes using the same assay.

Kelvin probe force microscopy of DNA-capped nanoparticles for single-nucleotide polymorphism detection.

Kelvin probe force microscopy (KPFM) is a robust toolkit for profiling the surface potential (SP) of biomolecular interactions between DNAs and/or proteins at the single molecule level. However, it has often suffered from background noise and low throughput due to instrumental or environmental constraints, which is regarded as limiting KPFM applications for detection of minute changes in the molecular structures such as single-nucleotide polymorphism (SNP). Here, we show KPFM imaging of DNA-capped nanoparticles (DCNP) that enables SNP detection of the BRCA1 gene owing to sterically well-adjusted DNA-DNA interactions that take place within the confined spaces of DCNP. The average SP values of DCNP interacting with BRCA1 SNP were found to be lower than the DCNP reacting with normal (non-mutant) BRCA1 gene. We also demonstrate that SP characteristics of DCNP with different substrates (e.g., Au, Si, SiO2, and Fe) provide us with a chance to attenuate or augment the SP signal of DCNP without additional enhancement of instrumentation capabilities.

Dim-light photoreceptor of chub mackerel Scomber japonicus and the photoresponse upon illumination with LEDs of different wavelengths.

To study the absorption characteristics of rhodopsin, a dim-light photoreceptor, in chub mackerel (Scomber japonicus) and the relationship between light wavelengths on the photoresponse, the rod opsin gene was cloned into an expression vector, pMT4. Recombinant opsin was transiently expressed in COS-1 cells and reconstituted with 11-cis-retinal. Cells containing the regenerated rhodopsin were solubilized and subjected to UV/Vis spectroscopic analysis in the dark and upon illumination. Difference spectra from the lysates indicated an absorption maximum of mackerel rhodopsin around 500 nm. Four types of light-emitting diode (LED) modules with different wavelengths (red, peak 627 nm; cyan, 505 nm; blue, 442 nm; white, 447 + 560 nm) were constructed to examine their effects on the photoresponse in chub mackerel. Behavioral responses of the mackerels, including speed and frequencies acclimated in the dark and upon LED illumination, were analyzed using an underwater acoustic camera. Compared to an average speed of 22.25 ± 1.57 cm/s of mackerel movement in the dark, speed increased to 22.97 ± 0.29, 24.66 ± 1.06, 26.28 ± 2.28, and 25.19 ± 1.91 cm/s upon exposure to red, blue, cyan, and white LEDs, respectively. There were increases of 103.48 ± 1.58, 109.37 ± 5.29, 118.48 ± 10.82, and 109.43 ± 3.92 %, respectively, in the relative speed of the fishes upon illumination with red, blue, cyan, and white LEDs compared with that in the dark (set at 100 %). Similar rate of wavelength-dependent responses was observed in a frequency analysis. These results indicate that an LED emitting a peak wavelength close to an absorption maximum of rhodopsin is more effective at eliciting a response to light.

Self-assembled amyloid fibrils with controllable conformational heterogeneity.

Amyloid fibrils are a hallmark of neurodegenerative diseases and exhibit a conformational diversity that governs their pathological functions. Despite recent findings concerning the pathological role of their conformational diversity, the way in which the heterogeneous conformations of amyloid fibrils can be formed has remained elusive. Here, we show that microwave-assisted chemistry affects the self-assembly process of amyloid fibril formation, which results in their conformational heterogeneity. In particular, microwave-assisted chemistry allows for delicate control of the thermodynamics of the self-assembly process, which enabled us to tune the molecular structure of ß-lactoglobulin amyloid fibrils. The heterogeneous conformations of amyloid fibrils, which can be tuned with microwave-assisted chemistry, are attributed to the microwave-driven thermal energy affecting the electrostatic interaction during the self-assembly process. Our study demonstrates how microwave-assisted chemistry can be used to gain insight into the origin of conformational heterogeneity of amyloid fibrils as well as the design principles showing how the molecular structures of amyloid fibrils can be controlled.

Sound packing DNA: packing open circular DNA with low-intensity ultrasound.

Supercoiling DNA (folding DNA into a more compact molecule) from open circular forms requires significant bending energy. The double helix is coiled into a higher order helix form; thus it occupies a smaller footprint. Compact packing of DNA is essential to improve the efficiency of gene delivery, which has broad implications in biology and pharmaceutical research. Here we show that low-intensity pulsed ultrasound can pack open circular DNA into supercoil form. Plasmid DNA subjected to 5.4 mW/cm(2) intensity ultrasound showed significant (p-values <0.001) supercoiling compared to DNA without exposure to ultrasound. Radiation force induced from ultrasound and dragging force from the fluid are believed to be the main factors that cause supercoiling. This study provides the first evidence to show that low-intensity ultrasound can directly alter DNA topology. We anticipate our results to be a starting point for improved non-viral gene delivery.

Nanomechanical characterization of chemical interaction between gold nanoparticles and chemical functional groups.

We report on how to quantify the binding affinity between a nanoparticle and chemical functional group using various experimental methods such as cantilever assay, PeakForce quantitative nanomechanical property mapping, and lateral force microscopy. For the immobilization of Au nanoparticles (AuNPs) onto a microscale silicon substrate, we have considered two different chemical functional molecules of amine and catecholamine (here, dopamine was used). It is found that catecholamine-modified surface is more effective for the functionalization of AuNPs onto the surface than the amine-modified surface, which has been shown from our various experiments. The dimensionless parameter (i.e., ratio of binding affinity) introduced in this work from such experiments is useful in quantitatively depicting such binding affinity, indicating that the binding affinity and stability between AuNPs and catecholamine is approximately 1.5 times stronger than that between amine and AuNPs. Our study sheds light on the experiment-based quantitative characterization of the binding affinity between nanomaterial and chemical groups, which will eventually provide an insight into how to effectively design the functional material using chemical groups.