Predicting Serotonin Detection with DNA-Carbon Nanotube Sensors across Multiple Spectral Wavelengths.

Owing to the value of DNA-wrapped single-walled carbon nanotube (SWNT)-based sensors for chemically specific imaging in biology, we explore machine learning (ML) predictions DNA-SWNT serotonin sensor responsivity as a function of DNA sequence based on the whole SWNT fluorescence spectra. Our analysis reveals the crucial role of DNA sequence in the binding modes of DNA-SWNTs to serotonin, with a smaller influence of SWNT chirality. Regression ML models trained on existing data sets predict the change in the fluorescence emission in response to serotonin, ΔF/F, at over a hundred wavelengths for new DNA-SWNT conjugates, successfully identifying some high- and low-response DNA sequences. Despite successful predictions, we also show that the finite size of the training data set leads to limitations on prediction accuracy. Nevertheless, incorporating entire spectra into ML models enhances prediction robustness and facilitates the discovery of novel DNA-SWNT sensors. Our approaches show promise for identifying new chemical systems with specific sensing response characteristics, marking a valuable advancement in DNA-based system discovery.

Directed Evolution of Near-Infrared Serotonin Nanosensors with Machine Learning-Based Screening.

In this study, we employed a novel approach to improve the serotonin-responsive ssDNA-wrapped single-walled carbon nanotube (ssDNA-SWCNT) nanosensors, combining directed evolution and machine learning-based prediction. Our iterative optimization process is aimed at the sensitivity and selectivity of ssDNA-SWCNT nanosensors. In the three rounds for higher serotonin sensitivity, we substantially improved sensitivity, achieving a remarkable 2.5-fold enhancement in fluorescence response compared to the original sequence. Following this, we directed our efforts towards selectivity for serotonin over dopamine in the two rounds. Despite the structural similarity between these neurotransmitters, we achieved a 1.6-fold increase in selectivity. This innovative methodology, offering high-throughput screening of mutated sequences, marks a significant advancement in biosensor development. The top-performing nanosensors, N2-1 (sensitivity) and L1-14 (selectivity) present promising reference sequences for future studies involving serotonin detection.

Lysophosphatidic acid induces proliferation and osteogenic differentiation of human dental pulp stem cell through lysophosphatidic acid receptor 3/extracellular signal-regulated kinase signaling axis.

Background/purpose: Human dental pulp stem cells (hDPSCs) possess excellent proliferative and osteogenic differentiation potentials. This study aimed to elucidate the role of lysophosphatidic acid (LPA) signaling in the proliferation and osteogenic differentiation of hDPSCs. Materials and methods: hDPSCs were treated with LPA and proliferation was measured using the cell counting kit-8 assay. Following the osteogenic differentiation of hDPSCs using osteogenic medium in the presence or absence of LPA, alkaline phosphatase (ALP) staining, ALP activity measurements, and RT-qPCR were performed to analyze the osteoblast differentiation. Small interfering RNA (siRNA)-mediated LPAR3 silencing and extracellular signal-regulated (ERK)/mitogen-activated protein (MAP) kinase inhibitors were used to elucidate the molecular mechanisms underlying LPA-induced proliferation and differentiation of hDPSCs. Results: LPA treatment significantly induced proliferation and osteogenic differentiation of hDPSCs. The depletion of LPAR3 expression by LPAR3-speicifc siRNA in hDPSCs diminished LPA-induced proliferation and osteogenic differentiation. The LPAR3-mediated proliferation and osteogenic differentiation of hDPSCs in response to LPA were significantly suppressed by U0126, a selective inhibitor of ERK. Conclusion: These findings suggest that LPA induces the proliferation and osteogenic differentiation of hDPSCs via LPAR3-ERK-dependent pathways.

Contrast Agents for Photoacoustic Imaging: A Review Focusing on the Wavelength Range.

Photoacoustic imaging using endogenous chromophores as a contrast has been widely applied in biomedical studies owing to its functional imaging capability at the molecular level. Various exogenous contrast agents have also been investigated for use in contrast-enhanced imaging and functional analyses. This review focuses on contrast agents, particularly in the wavelength range, for use in photoacoustic imaging. The basic principles of photoacoustic imaging regarding light absorption and acoustic release are introduced, and the optical characteristics of tissues are summarized according to the wavelength region. Various types of contrast agents, including organic dyes, semiconducting polymeric nanoparticles, gold nanoparticles, and other inorganic nanoparticles, are explored in terms of their light absorption range in the near-infrared region. An overview of the contrast-enhancing capacity and other functional characteristics of each agent is provided to help researchers gain insights into the development of contrast agents in photoacoustic imaging.

Discovery of DNA-Carbon Nanotube Sensors for Serotonin with Machine Learning and Near-infrared Fluorescence Spectroscopy.

DNA-wrapped single walled carbon nanotube (SWNT) conjugates have distinct optical properties leading to their use in biosensing and imaging applications. A critical limitation in the development of DNA-SWNT sensors is the current inability to predict unique DNA sequences that confer a strong analyte-specific optical response to these sensors. Here, near-infrared (nIR) fluorescence response data sets for ∼100 DNA-SWNT conjugates, narrowed down by a selective evolution protocol starting from a pool of ∼1010 unique DNA-SWNT candidates, are used to train machine learning (ML) models to predict DNA sequences with strong optical response to neurotransmitter serotonin. First, classifier models based on convolutional neural networks (CNN) are trained on sequence features to classify DNA ligands as either high response or low response to serotonin. Second, support vector machine (SVM) regression models are trained to predict relative optical response values for DNA sequences. Finally, we demonstrate with validation experiments that integrating the predictions of ensembles of the highest quality neural network classifiers (convolutional or artificial) and SVM regression models leads to the best predictions of both high and low response sequences. With our ML approaches, we discovered five DNA-SWNT sensors with higher fluorescence intensity response to serotonin than obtained previously. Overall, the explored ML approaches, shown to predict useful DNA sequences, can be used for discovery of DNA-based sensors and nanobiotechnologies.

Extraction of Viral Nucleic Acids with Carbon Nanotubes Increases SARS-CoV-2 Quantitative Reverse Transcription Polymerase Chain Reaction Detection Sensitivity.

The global SARS-CoV-2 coronavirus pandemic has led to a surging demand for rapid and efficient viral infection diagnostic tests, generating a supply shortage in diagnostic test consumables including nucleic acid extraction kits. Here, we develop a modular method for high-yield extraction of viral single-stranded nucleic acids by using "capture" ssDNA sequences attached to carbon nanotubes. Target SARS-CoV-2 viral RNA can be captured by ssDNA-nanotube constructs via hybridization and separated from the liquid phase in a single-tube system with minimal chemical reagents, for downstream quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection. This nanotube-based extraction method enables 100% extraction yield of target SARS-CoV-2 RNA from phosphate-buffered saline in comparison to ∼20% extraction yield when using a commercial silica-column kit. Notably, carbon nanotubes enable extraction of nucleic acids directly from 50% human saliva with a similar efficiency as achieved with commercial DNA/RNA extraction kits, thereby bypassing the need for further biofluid purification and avoiding the use of commercial extraction kits. Carbon nanotube-based extraction of viral nucleic acids facilitates high-yield and high-sensitivity identification of viral nucleic acids such as the SARS-CoV-2 viral genome with a reduced reliance on reagents affected by supply chain obstacles.

Zwitterion-Coated Colloidal Magnetic Nanoparticle Clusters for Reduced Nonspecific Adsorption of Biomolecules.

This paper demonstrates fabrication of silica-shell-coated magnetic nanoparticle clusters (SMNCs) and subsequent surface engineering of SMNCs to produce surface-modified SMNCs that have zwitterionic and primary amine ligands (SMNC-ZW/Am). SMNC-ZW/Am was passivated by zwitterionic ligands for improved colloidal stability and reduced nonspecific adsorption and by primary amine ligands for facilitated conjugation with biomolecules. Hydrodynamic (HD) size and zeta potential of SMNC-ZW/Am could be flexibly tuned by controlling the relative amounts of zwitterionic and primary amine ligands. SMNC-ZW/Am had higher colloidal stability in high salt concentration and broad pH range than did bare SMNC. Nonspecific adsorption with biomolecules onto SMNC-ZW/Am surface was significantly suppressed by the zwitterionic ligands. The facile bioconjugation capability of SWNC-ZW/Am enabled conjugation of biotin and antibody to the SWNC-ZW/Am surface. Biomolecule-conjugated SMNC-ZW/Am showed specific binding affinity to streptavidin and Salmonella bacteria, with reduced nonspecific adsorption; therefore, SWMC-ZW/Am has potential use as an antifouling nanosubstrate for separation and bioanalysis.

Advances in engineering near-infrared luminescent materials.

Near-infrared (NIR) luminescent materials have emerged as a growing field of interest, particularly for imaging and optics applications in biology, chemistry, and physics. However, the development of materials for this and other use cases has been hindered by a range of issues that prevents their widespread use beyond benchtop research. This review explores emerging trends in some of the most promising NIR materials and their applications. In particular, we focus on how a more comprehensive understanding of intrinsic NIR material properties might allow researchers to better leverage these traits for innovative and robust applications in biological and physical sciences.

Rapid SARS-CoV-2 Spike Protein Detection by Carbon Nanotube-Based Near-Infrared Nanosensors.

To effectively track and eliminate COVID-19, it is critical to develop tools for rapid and accessible diagnosis of actively infected individuals. Here, we introduce a single-walled carbon nanotube (SWCNT)-based optical sensing approach toward this end. We construct a nanosensor based on SWCNTs noncovalently functionalized with ACE2, a host protein with high binding affinity for the SARS-CoV-2 spike protein. The presence of the SARS-CoV-2 spike protein elicits a robust, 2-fold nanosensor fluorescence increase within 90 min of spike protein exposure. We characterize the nanosensor stability and sensing mechanism and passivate the nanosensor to preserve sensing response in saliva and viral transport medium. We further demonstrate that these ACE2-SWCNT nanosensors retain sensing capacity in a surface-immobilized format, exhibiting a 73% fluorescence turn-on response within 5 s of exposure to 35 mg/L SARS-CoV-2 virus-like particles. Our data demonstrate that ACE2-SWCNT nanosensors can be developed into an optical tool for rapid SARS-CoV-2 detection.

Rapid SARS-CoV-2 Detection by Carbon Nanotube-Based Near-Infrared Nanosensors.

To effectively track and eliminate COVID-19, it is critical to develop tools for rapid and accessible diagnosis of actively infected individuals. Here, we introduce a single-walled carbon nanotube (SWCNT)-based optical sensing approach towards these ends. We construct a nanosensor based on SWCNTs noncovalently functionalized with ACE2, a host protein with high binding affinity for the SARS-CoV-2 spike protein. Presence of the SARS-CoV-2 spike protein elicits a robust, two-fold nanosensor fluorescence increase within 90 min of spike protein exposure. We characterize the nanosensor stability and sensing mechanism, and passivate the nanosensor to preserve sensing response in saliva and viral transport medium. We further demonstrate that these ACE2-SWCNT nanosensors retain sensing capacity in a surface-immobilized format, exhibiting a 73% fluorescence turn-on response within 5 s of exposure to 35 mg/L SARS-CoV-2 virus-like particles. Our data demonstrate that ACE2-SWCNT nanosensors can be developed into an optical tool for rapid SARS-CoV-2 detection.

Ultrasound Standing Wave-Based Cell-to-liquid Separation for Measuring Viscosity and Aggregation of Blood Sample.

When quantifying mechanical properties of blood samples flowing in closed fluidic circuits, blood samples are collected at specific intervals. Centrifugal separation is considered as a required procedure for preparing blood samples. However, the use of centrifuge is associated with several issues, including the potential for red blood cell (RBC) lysis, clotting activation, and RBC adhesions in the tube. In this study, an ultrasonic transducer is employed to separate RBCs or diluent from blood sample. The ultrasonic radiation force is much smaller than the centrifugal force acting in centrifuge, it can avoid critical issues occurring under centrifuge. Then, the RBC aggregation and blood viscosity of the blood sample are obtained using the microfluidic technique. According to the numerical results, ultrasonic transducers exhibited a maximum quality factor at an excitation frequency of 2.1 MHz. Periodic pattern of acoustic pressure fields were visualized experimentally as a column mode. The half wavelength obtained was as 0.5 λ = 0.378 ± 0.07 mm. The experimental results agreed with the analytical estimation sufficiently. An acoustic power of 2 W was selected carefully for separating RBCs or diluent from various blood samples (i.e., Hct = 20% ~ 50%; diluent: plasma, 1x phosphate-buffered saline (PBS), and dextran solution). The present method was employed to separate fixed blood samples which tended to stack inside the tube while using the centrifuge. Fixed RBCs were collected easily with an ultrasonic transducer. After various fixed blood samples with different base solutions (i.e., glutaraldehyde solution, 1x PBS, and dextran solution) were prepared using the present method, RBC aggregation and the viscosity of the blood sample are successfully obtained. In the near future, the present method will be integrated into ex vivo or in vitro fluidic circuit for measuring multiple mechanical properties of blood samples for a certain longer period.

Graphene Quantum Dot Oxidation Governs Noncovalent Biopolymer Adsorption.

Graphene quantum dots (GQDs) are an allotrope of carbon with a planar surface amenable to functionalization and nanoscale dimensions that confer photoluminescence. Collectively, these properties render GQDs an advantageous platform for nanobiotechnology applications, including optical biosensing and delivery. Towards this end, noncovalent functionalization offers a route to reversibly modify and preserve the pristine GQD substrate, however, a clear paradigm has yet to be realized. Herein, we demonstrate the feasibility of noncovalent polymer adsorption to GQD surfaces, with a specific focus on single-stranded DNA (ssDNA). We study how GQD oxidation level affects the propensity for polymer adsorption by synthesizing and characterizing four types of GQD substrates ranging ~60-fold in oxidation level, then investigating noncovalent polymer association to these substrates. Adsorption of ssDNA quenches intrinsic GQD fluorescence by 31.5% for low-oxidation GQDs and enables aqueous dispersion of otherwise insoluble no-oxidation GQDs. ssDNA-GQD complexation is confirmed by atomic force microscopy, by inducing ssDNA desorption, and with molecular dynamics simulations. ssDNA is determined to adsorb strongly to no-oxidation GQDs, weakly to low-oxidation GQDs, and not at all for heavily oxidized GQDs. Finally, we reveal the generality of the adsorption platform and assess how the GQD system is tunable by modifying polymer sequence and type.

Comparison of antioxidant capacity of 4-vinylguaiacol with catechin and ferulic acid in oil-in-water emulsion.

The product of ferulic acid decarboxylation, 4-vinylguaiacol (4-VG), is an important antioxidant and is reported to have an antioxidant capacity comparable to α-tocopherol. In this study, evaluation on antioxidant capacities of ferulic acid, catechin, and 4-VG was performed when 200 ppm of each compound was added in a 10% O/W emulsion for 50 days. Peroxide value (POV) results of the O/W emulsion containing 4-VG were noteworthy. The POV was 1.9 meq/L of emulsion after 29 days, which was no different to the initial value (day 0). Even when the oxidation was allowed to advance to day 50, the POV remained at 2.2 meq/L of emulsion, representing only a tiny increase relative to the initial value on day 0. 1H-NMR results also showed that the lowest conjugated forms and no aldehydes were detected in emulsion of 4-VG stored for 50 days, proving the excellent antioxidant capacity in the O/W emulsion.

High-throughput evolution of near-infrared serotonin nanosensors.

Imaging neuromodulation with synthetic probes is an emerging technology for studying neurotransmission. However, most synthetic probes are developed through conjugation of fluorescent signal transducers to preexisting recognition moieties such as antibodies or receptors. We introduce a generic platform to evolve synthetic molecular recognition on the surface of near-infrared fluorescent single-wall carbon nanotube (SWCNT) signal transducers. We demonstrate evolution of molecular recognition toward neuromodulator serotonin generated from large libraries of ~6.9 × 1010 unique ssDNA sequences conjugated to SWCNTs. This probe is reversible and produces a ~200% fluorescence enhancement upon exposure to serotonin with a K d = 6.3 µM, and shows selective responsivity over serotonin analogs, metabolites, and receptor-targeting drugs. Furthermore, this probe remains responsive and reversible upon repeat exposure to exogenous serotonin in the extracellular space of acute brain slices. Our results suggest that evolution of nanosensors could be generically implemented to develop other neuromodulator probes with synthetic molecular recognition.

Chemometric Approaches for Developing Infrared Nanosensors To Image Anthracyclines.

Generation, identification, and validation of optical probes to image molecular targets in a biological milieu remain a challenge. Synthetic molecular recognition approaches leveraging the intrinsic near-infrared fluorescence of single-walled carbon nanotubes are promising for long-term biochemical imaging in tissues. However, generation of nanosensors for selective imaging of molecular targets requires a heuristic approach. Here, we present a chemometric platform for rapidly screening libraries of candidate single-walled carbon nanotube nanosensors against biochemical analytes to quantify the fluorescence response to small molecules, including vitamins, neurotransmitters, and chemotherapeutics. We further show this method can be applied to identify biochemical analytes that selectively modulate the intrinsic near-infrared fluorescence of candidate nanosensors. Chemometric analysis thus enables identification of nanosensor-analyte "hits" and also nanosensor fluorescence signaling modalities such as wavelength shifts that are optimal for translation to biological imaging. Through this approach, we identify and characterize a nanosensor for the chemotherapeutic anthracycline doxorubicin (DOX), which provides a ≤17 nm fluorescence red-shift and exhibits an 8 µM limit of detection, compatible with peak circulatory concentrations of doxorubicin common in therapeutic administration. We demonstrate the selectivity of this nanosensor over dacarbazine, a chemotherapeutic commonly co-injected with doxorubicin. Lastly, we establish nanosensor tissue compatibility for imaging of doxorubicin in muscle tissue by incorporating nanosensors into the mouse hindlimb and measuring the nanosensor response to exogenous DOX administration. Our results motivate chemometric approaches to nanosensor discovery for chronic imaging of drug partitioning into tissues and toward real-time monitoring of drug accumulation.

Multiplexed In Vivo Imaging Using Size-Controlled Quantum Dots in the Second Near-Infrared Window.

PbS/CdS core/shell quantum dots (QDs) that emit at the second near-infrared (NIR-II, 1000-1700 nm) window are synthesized. The PbS seed size and CdS shell thicknesses are carefully controlled to produce bright and narrow fluorescence that are suitable for multiplexing. A polymer encapsulation yields polymer-encapsulated NIR-II QDs (PQDs), which provides the QDs with long-term fluorescence stability over a week in biological media. Exploiting the simple bioconjugation capability of PQDs, folic acids are conjugated to PQDs that can efficiently label folate receptor overexpressing cell lines. The PQDs afford multiplexed and nearly real-time longitudinal whole-body in vivo imaging. Two NIR-II QD probes are prepared: folic acid-conjugated PQDs (FA-PQDs) emitting at 1280 nm and unconjugated PQDs emitting at 1080 nm. The two PQDs are engineered to have compact and similar hydrodynamic sizes. A mixture of the folic acid-conjugated PQD and unconjugated PQDs is injected intravenously into a tumor-xenografted mouse, and the signals from them are monitored. This NIR-II whole-body imaging with the two PQDs provides precise evaluation of the active ligand-assisted tumor-targeting capability of the FA-PQD probe because the hydrodynamic size control of the two PQDs effectively eliminates effects from the size-dependent accumulations by permeations and retentions in tumors.

Heterojunction Area-Controlled Inorganic Nanocrystal Solar Cells Fabricated Using Supra-Quantum Dots.

A supra-quantum dot (SQD) is a three-dimensional structure formed by the attachment of quantum dots. The SQDs have sizes of tens of nanometer and they maintain the characteristics of the individual quantum dots fairly well. Moreover, their sizes and elemental compositions can be tuned precisely. On the basis of their unique features, in this work, SQDs are used as constituents of the interpenetrating photoactive layers of inorganic nanocrystal p-n heterojunction solar cells to control the p-type and n-type domain sizes (i.e., p-n heterojunction areas) for optimizing the charge-carrier collection. SQD-containing p-n heterojunction solar cells exhibit improved charge transport and thereby higher power conversion efficiency (PCE) (3.03%) owing to their intermediate p-type and n-type domain sizes, which are between those of a bilayer nanorod p-n heterojunction solar cell (PCE: 1.21%) and an interpenetrating nanorod p-n heterojunction solar cell (PCE: 2.40%). This work demonstrates that the self-assembly of nanoscale materials can be utilized for tailoring the spatial distributions of charge carriers, which is beneficial for obtaining an enhanced device performance.

Antioxidant capacity of decarboxylated rice bran extract in bulk oil.

Decarboxylation of ferulic acid would increase the solubility in oils. Rice bran extract (RBE) containing 29 mg ferulic acid/g RBE was decarboxylated to obtain decarboxylated rice bran extract (DRBE), and its antioxidant capacity in oil system was studied. After addition of DRBE (500 ppm), oxidation was monitored for 20 days at 60 °C under the dark. To compare the oxidation degree, 500 ppm of ferulic acid and well-known lipid soluble antioxidant, α-tocopherol, were used. Contents of conjugated dienes and aldehydes were measured using 1H NMR as well as peroxide value (POV). On 7 days of oxidation, DRBE (539.0 meq/kg oil) showed lower POV than the control (819.7 meq/kg oil). Also, contents of total conjugated form and aldehydes were 194.60, and 5.94 mmol/L oil, which were lower than those of control (323.63 and 15.94 mmol/L oil). However, after 10 days of oxidation, antioxidant capacity of DRBE was not observed.

Cancer-Microenvironment-Sensitive Activatable Quantum Dot Probe in the Second Near-Infrared Window.

Recent technological advances have expanded fluorescence (FL) imaging into the second near-infrared region (NIR-II; wavelength = 1000-1700 nm), providing high spatial resolution through deep tissues. However, bright and compact fluorophores are rare in this region, and sophisticated control over NIR-II probes has not been fully achieved yet. Herein, we report an enzyme-activatable NIR-II probe that exhibits FL upon matrix metalloprotease activity in tumor microenvironment. Bright and stable PbS/CdS/ZnS core/shell/shell quantum dots (QDs) were synthesized as a model NIR-II fluorophore, and activatable modulators were attached to exploit photoexcited electron transfer (PET) quenching. The quasi type-II QD band alignment allowed rapid and effective FL modulations with the compact surface ligand modulator that contains methylene blue PET quencher. The modulator was optimized to afford full enzyme accessibility and high activation signal surge upon the enzyme activity. Using a colon cancer mouse model, the probe demonstrated selective FL activation at tumor sites with 3-fold signal enhancement in 10 min. Optical phantom experiments confirmed the advantages of the NIR-II probe over conventional dyes in the first near-infrared region.

"Smart" gold nanoparticles for photoacoustic imaging: an imaging contrast agent responsive to the cancer microenvironment and signal amplification via pH-induced aggregation.

'Smart' gold nanoparticles can respond to mild acidic environments, rapidly form aggregates, and shift the absorption to red and near-infrared. They were used as a photoacoustic imaging agent responsive to the cancer microenvironment, and have demonstrated the cancer-specific accumulation at the cellular level and an amplified signal which is twice higher than the control in vivo.