Real-Time Monitoring of the Evaporation and Fission of Electrospray-Ionized Polystyrene Beads and Bacterial Pellets at Elevated Temperatures.

This study uses real-time monitoring, at microsecond time scales, with a charge-sensing particle detector to investigate the evaporation and fission processes of methanol/micrometer-sized polystyrene beads (PS beads) droplets and bacterial particles droplets generated via electrospray ionization (ESI) under elevated temperatures. By incrementally raising capillary temperatures, the solvent, such as methanol on 0.75 µm PS beads, experiences partial evaporation. Further temperature increase induces fission, and methanol molecules continue to evaporate until PS ions are detected after this range. Similar partial evaporation is observed on 3 µm PS beads. However, the shorter period of the fission temperature range is necessary compared to 0.75 µm PS beads. For the spherical-shaped bacterium, Staphylococcus aureus, the desolvation process shows a similar fission period as compared to 0.75 µm PS beads. Comparably, the rod-shaped bacteria, Escherichia coli EC11303, and E. coli strain W have shorter fission periods than S. aureus. This research provides insights into the evaporation and fission mechanisms of ESI droplets containing different sizes and shapes of micrometer-sized particles, contributing to a better understanding of gaseous macroion formation.

Ionization of Submicrometer-Sized Particles by Laser-Induced Radiofrequency Plasma for Mass Spectrometric Analysis.

A laser-induced rf plasma (LIRFP) ion source was developed to ionize submicrometer-sized particles for the first time. The LIRFP ion source can increase the charge of those particles to several thousand charges via charge exchange reactions so that those particles can be trapped and analyzed with a charge detection quadrupole ion trap-mass spectrometer (CD QIT-MS). Different reagent gases for charge exchange reaction were investigated, viz. argon, nitrogen, oxygen, methane, helium, krypton, xenon, argon/methane (with ratios of 10:1 and 2:1), argon/nitrogen (with a ratio of 1:1), nitrogen/oxygen (10:1), krypton/methane (10:1), and air. The average charge of 0.75 µm polystyrene particles could reach 1631 using an argon/methane mixture with a ratio of ∼10:1. The average charges for freeze-dried Escherichia coli EC11303, Escherichia coli strain W, and Staphylococcus aureus were 842, 1112, and 971, respectively, with a mass-to-charge ratio ( m/ z) range from 107 to 108; and the average masses were 3.5 × 1010 Da, 6.0 × 1010 Da, and 5.6 × 1010 Da, respectively. The average mass and charge of the vaccinia virus were ∼9.1 × 109 Da and ∼708 with a m/ z of ∼107. This LIRFP CD QIT-MS method was rapid with only 20 min for each sample measurement.

Ambient aerodynamic desorption/ionization method for microparticle mass measurement.

An ambient desorption/ionization method, named aerodynamic desorption (AD), was proposed for the in situ rapid mass measurement of microparticles. The AD method exploited the discontinuous atmospheric pressure interface (DAPI) to generate a pulsed airflow, which was used to desorb the microparticles under atmospheric pressure. Various microparticles, e.g., bacteria, cell, polystyrene, synthetic diamond, and silica particles, with different size and surface component were successfully desorbed. Similar to that in the conventional laser-induced acoustic desorption (LIAD) method, these microparticles were desorbed as precharged ions in the AD process and the charge number was largely relevant to the particle size. However, compared with LIAD, the sensitivity of the AD method was higher. A lower concentration of particles was required for the analysis. In addition, the construction and sampling process of AD source were much simpler. All types of liquid, solid, or/and gaseous samples can be directly sampled under ambient condition. As a demonstration of this AD method, the in situ mass analysis of red blood cells (RBCs) and E. coli bacteria were carried out using a homemade ambient AD mass spectrometer consisting of AD source, QIT mass analyzer, and charge detector. Their mass and mass distributions were obtained successfully.

A Novel Endodontic Approach in Removing Smear Layer Using Nano and Submicron Diamonds with Intracanal Oscillation Irrigation.

Sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic acid (EDTA) are commonly recommended for effectively removing organic and inorganic components in the smear layer. This layer is found on root canal walls after root canal instrumentation. However, high-concentration EDTA reduces the strength of dentin and the dissolution efficacy of organic substances in NaOCl solution. The objective of this study was to investigate the efficacy of applying nano and submicron diamonds in irrigation solutions with sonic and ultrasonic oscillation for removing the smear layer during endodontic treatment. Extracted single-rooted human teeth were instrumented with ProTaper® Gold (Dentsply Sirona) nickel-titanium rotary instruments. Subsequently, each canal was irrigated with 3% NaOCl, 17% EDTA, distilled water, and 10-1000 nm-sized nano and submicron diamond irrigation solutions, respectively. Sonic and ultrasonic instruments were compared for oscillating the irrigation solutions. The teeth were processed for scanning electron microscopy to observe the efficiency of smear layer removal on the canal walls. Our results indicated that diamond sizes of 50 nm and above irrigation solutions showed significant effectiveness in removing the smear layer following the oscillation of sonic instruments for 10 s. Ultrasonic assisted 500 nm and 1000 nm diamond solutions significantly differed from the other diamond-sized solution in their ability to remove the smear layer. These results suggest that sonic and ultrasonic oscillation with specific sizes of nano and submicron diamond irrigation solution can be used as an alternative approach to removing the smear layer during endodontic treatment. The potential clinical application of root canal treatments can be expected.

Fluorescent nanodiamond-based spin-enhanced lateral flow immunoassay for detection of SARS-CoV-2 nucleocapsid protein and spike protein from different variants.

SARS-CoV-2 viruses, responsible for the COVID-19 pandemic, continues to evolve into new mutations, which poses a significant threat to public health. Current testing methods have some limitations, such as long turnaround times, high costs, and professional laboratory requirements. In this report, the novel Spin-Enhanced Lateral Flow Immunoassay (SELFIA) platform and fluorescent nanodiamond (FND) reporter were utilized for the rapid detection of SARS-CoV-2 nucleocapsid and spike antigens from different variants, including wild-type (Wuhan-1), Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529). The SARS-CoV-2 antibodies were conjugated with FND via nonspecific binding, enabling the detection of SARS-CoV-2 antigens via both direct and competitive SELFIA format. Direct SELFIA was performed by directly adding the SARS-CoV-2 antibodies-conjugated FND on the antigens-immobilized nitrocellulose (NC) membrane. Conversely, the SARS-CoV-2 antigen-containing sample was first incubated with the antibodies-conjugated FND, and then dropped on the antigen-immobilized NC membrane to carry out the competitive SELFIA. The results suggested that S44F anti-S IgG antibody can be efficiently used for the detection of wild-type, Alpha, Delta, and Omicron variants spike antigens. Findings were comparable in direct SELFIA, competitive SELFIA, and ELISA. A detection limit of 1.94, 0.77, 1.14, 1.91, and 1.68 ng/mL can be achieved for SARS-CoV-2 N protein, wild-type, Alpha, Delta, and Omicron S proteins, respectively, via competitive SELFIA assay. These results suggest that a direct SELFIA assay can be used for antibody/antigen pair screening in diagnosis development, while the competitive SELFIA assay can serve as an accurate quantitative diagnostic tool. The simplicity and rapidity of the SELFIA platform were demonstrated, which can be leveraged in the detection of other infectious diseases in the near future.

Receptor-mediated cellular uptake of folate-conjugated fluorescent nanodiamonds: a combined ensemble and single-particle study.

Fluorescent nanodiamonds (FNDs) are nontoxic and photostable nanomaterials, ideal for long-term in vivo imaging applications. This paper reports that FNDs with a size of approximately 140 nm can be covalently conjugated with folic acid (FA) for receptor-mediated targeting of cancer cells at the single-particle level. The conjugation is made by using biocompatible polymers, such as polyethylene glycol, as crosslinked buffer layers. Ensemble-averaged measurements with flow cytometry indicate that more than 50% of the FA-conjugated FND particles can be internalized by the cells (such as HeLa cells) through receptor-mediated endocytosis, as confirmed by competitive inhibition assays. Confocal fluorescence microscopy reveals that these FND particles accumulate in the perinuclear region. The absolute number of FNDs internalized by HeLa cells after 3 h of incubation at a particle concentration of 10 microg mL(-1) is in the range of 100 particles per cell. The receptor-mediated uptake process is further elucidated by single-particle tracking of 35-nm FNDs in three dimensions and real time during the endocytosis.

The biocompatibility of fluorescent nanodiamonds and their mechanism of cellular uptake.

The labeling of cells with fluorescent nanoparticles is promising for various biomedical applications. The objective of this study is to evaluate the biocompatibility and the mechanism of the cellular uptake of fluorescent nanodiamonds (FNDs) in cancer cells (HeLa) and pre-adipocytes (3T3-L1). With flow cytometry and the use of a battery of metabolic and cytoskeletal inhibitors, we found that the mechanism of the FND uptake in both cells is by energy-dependent clathrin-mediated endocytosis. In addition, the surface charge of FND influences its cellular uptake, as the uptake of poly-L-lysine-coated FNDs is better than that of oxidative-acid-purified FNDs at the same concentration in regular medium with or without serum. We also confirm that the proliferative potential of FND-treated and untreated cells does not exhibit any significant differences when measured at bulk cultures, and more stringently at clonal cell density. Further biocompatibility studies indicate that the in vitro differentiation of 3T3-L1 pre-adipocytes and 489-2 osteoprogenitors is not affected by the FND treatment. Our results show that FNDs are biocompatible and ideal candidates for potential applications in human stem cell research.

Nanodiamond-supported silver nanoparticles as potent and safe antibacterial agents.

Since its discovery nearly a century ago, antibiotics has been one of the most effective methods in treating infectious diseases and limiting pathogen spread. However, pathogens often build up antibiotic resistance over time, leading to serious failure of the treatment. Silver nanoparticle (AgNP) is an appealing alternative, but successful treatment of the bacterial infection requires a plentiful supply of AgNP, which can negatively impact human health if people are excessively exposed to the particles. Here, we present a method to overcome this challenge by synthesizing nanodiamond-supported AgNP noncovalently conjugated with albumin molecules to achieve enhanced antibacterial activity and strengthened biocompatibility. Using Escherichia coli as a model bacterium, we found that the albumin-conjugated silver-diamond nanohybrids showed a long-term bactericidal effect after 36 days of the treatment at the AgNP concentration of 250 µg mL-1. Moreover, the toxicity of the nanohybrids to human cells (including human fibroblasts, lung adenocarcinoma epithelial cells, and breast adenocarcinoma cells) is low even at the particle concentration of 500 µg mL-1. The method provides a general and practical solution to the concerns of bacterial resistance against AgNP and issues associated with the size, shape, aggregation, and toxicity of AgNP are largely resolved. Finally, we demonstrate that the nanohybrids can be readily incorporated into natural polysaccharides (such as guar gum) to form three-in-one hydrogels, showing promising applications in nanomedicine.

Fluorescent nanodiamonds enable quantitative tracking of human mesenchymal stem cells in miniature pigs.

Cell therapy is a promising strategy for the treatment of human diseases. While the first use of cells for therapeutic purposes can be traced to the 19th century, there has been a lack of general and reliable methods to study the biodistribution and associated pharmacokinetics of transplanted cells in various animal models for preclinical evaluation. Here, we present a new platform using albumin-conjugated fluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) in miniature pigs by magnetic modulation. With this background-free detection technique and time-gated fluorescence imaging, we have been able to precisely determine the numbers as well as positions of the transplanted FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration. The method is applicable to single-cell imaging and quantitative tracking of human stem/progenitor cells in rodents and other animal models as well.

Nanodiamond-mediated drug delivery and imaging: challenges and opportunities.

INTRODUCTION: The field of nanoparticle-based therapeutic systems is rapidly expanding encompassing a wide variety of practices ranging from detection to diagnosis to treatment. Recently a great potential of nanodiamond (ND) particles as a multimodal imaging/therapy platform has been demonstrated. AREAS COVERED: This review describes a unique set of properties of ND particles attractive for drug delivery and imaging applications and highlights the most recent ND-based multimodal imaging/therapy approaches and related biocompatibility studies. The spectrum of major advancements includes marked improvements in tumor treatment efficacy and safety based on integration of ND with doxorubicin (DOX). Recent progress of ND-mediated drug delivery in orthopedic, dental and ophthalmic applications is also discussed. EXPERT OPINION: ND particles possess a unique set of properties attractive for drug delivery applications, including exceptional biocompatibility, large carrier capacity and versatile surface chemistry properties, which enhance drug binding and provide sustainable drug release. Other unique attributes of NDs embrace bright stable fluorescence based on crystallographic defects. A roadmap toward a clinical translation comprises identification of ND-therapeutic compounds that display marked improvements over clinical standards with respects to efficacy, safety and cost.

Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds.

Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphology and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphological alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker ß-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells.

The hemocompatibility of oxidized diamond nanocrystals for biomedical applications.

Low-dimensional carbon-based nanomaterials have recently received enormous attention for biomedical applications. However, increasing evidence indicates that they are cytotoxic and can cause inflammatory responses in the body. Here, we show that monocrystalline nanodiamonds (NDs) synthesized by high-pressure-high-temperature (HPHT) methods and purified by air oxidation and strong oxidative acid treatments have excellent hemocompatibility with negligible hemolytic and thrombogenic activities. Cell viability assays with human primary endothelial cells suggested that the oxidized HPHT-NDs (dimensions of 35-500 nm) are non-cytotoxic. No significant elevation of the inflammatory cytokine levels of IL-1ß and IL-6 was detected in mice after intravenous injection of the nanocrystals in vivo. Using a hindlimb-ischemia mouse model, we demonstrated that 35-nm NDs after covalent conjugation with polyarginine are useful as a drug delivery vehicle of heparin for prolonged anticoagulation treatment. The present study lays a solid foundation for further therapeutic applications of NDs in biomedicine.

The long-term stability and biocompatibility of fluorescent nanodiamond as an in vivo contrast agent.

Nanocarbon is a promising type of biomaterial for diagnostic and therapeutic applications. Fluorescent nanodiamond (FND) containing nitrogen-vacancy centers as built-in fluorophores is a new addition to the nanocarbon family. Here, we study the long-term stability and biocompatibility of 100-nm FNDs in rats through intraperitoneal injection over 5 months and develop the potential application of this biomaterial for sentinel lymph node mapping in a mouse model. From both in vivo and ex vivo fluorescence imaging as well as transmission electron microscopy, we found that the intradermally administered FND particles can be drained from the injection sites by macrophages and selectively accumulated in the axillary lymph nodes of the treated mice. Our measurements of water consumption, fodder consumption, body weight, and organ index showed no significant difference between control and FND-treated groups of the rats. Histopathological analysis of various tissues and organs indicated that FNDs are non-toxic even when a large quantity, up to 75 mg/kg body weight, of the particles was administered intraperitoneally to the living animals. With the properties of wide-ranging biocompatibility and perfect chemical and photophysical stability, FND is well suited for use as a contrast agent for long-term in vivo imaging.

Matrix-assisted laser desorption/ionization (MALDI) mechanism revisited.

Although matrix-assisted laser desorption/ionization (MALDI) was developed more than a decade ago and broad applications have been successfully demonstrated, detailed mechanism of MALDI is still not well understood. Two major models; namely photochemical ionization (PI) and cluster ionization (CI) mechanisms have been proposed to explain many of experimental results. With the photochemical ionization model, analyte ions are considered to be produced from a protonation or deprotonation process involving an analyte molecule colliding with a matrix ion in the gas phase. With the cluster ionization model, charged particles are desorbed with a strong photoabsorption by matrix molecules. Analyte ions are subsequently produced by desolvation of matrix from cluster ions. Nevertheless, many observations still cannot be explained by these two models. In this work, we consider a pseudo proton transfer process during crystallization as a primary mechanism for producing analyte ions in MALDI. We propose an energy transfer induced disproportionation (ETID) model to explain the observation of an equal amount of positive and negative ions produced in MALDI for large biomolecules. Some experimental results are used for comparisons of various models.