A fluorescent nanodiamond foundation for quantum sensing in cells

Free radicals play a major role in the aging process as well as a most diseases. However, we barely know anything about them. These tiny molecules have an extremely short lifespan and are difficult to measure, while their role in health related processes is considerable. Fluorescent nanodiamonds are very small diamonds which can shed a light on this research question. These diamonds emit a constant light in a controlled setting. This is possible due to a small defect in the structure of the diamond, which makes it fluorescent. When free radicals are present, the light changes, which allows measurement of the radicals. During my PhD I have laid the basis for these biological measurements. Not all cells automatically take up diamonds or the diamonds tend to aggregate in cellular medium. By changing the solutions in which we administer the diamonds, we can prevent these obstacles. I have also performed a very detailed analysis of the cellular response on diamond uptake. Conveniently, the cells hardly show any response to the diamond uptake, an important result for our future measurements. In addition, I have developed new ways of targeting the diamonds to specific places in the cells, to obtain location specific information. Finally I have determined the subcellular location of the diamonds using a new technique, based on integrated electron microscopy. During my work I have laid the foundation for promising cellular research of ageing and disease using fluorescent nanodiamonds

Improving surface and defect center chemistry of fluorescent nanodiamonds for imaging purposes-a review

Diamonds are widely used for jewelry owing to their superior optical properties accounting for their fascinating beauty. Beyond the sparkle, diamond is highly investigated in materials science for its remarkable properties. Recently, fluorescent defects in diamond, particularly the negatively charged nitrogen-vacancy (NV-) center, have gained much attention: The NV- center emits stable, nonbleaching fluorescence, and thus could be utilized in biolabeling, as a light source, or as a Forster resonance energy transfer donor. Even more remarkable are its spin properties: with the fluorescence intensity of the NV- center reacting to the presence of small magnetic fields, it can be utilized as a sensor for magnetic fields as small as the field of a single electron spin. However, a reproducible defect and surface and defect chemistry are crucial to all applications. In this article we review methods for using nanodiamonds for different imaging purposes. The article covers (1) dispersion of particles, (2) surface cleaning, (3) particle size selection and reduction, (4) defect properties, and (5) functionalization and attachment to nanostructures, e.g., scanning probe microscopy tips

The Response of HeLa Cells to Fluorescent NanoDiamond Uptake

Fluorescent nanodiamonds are promising probes for nanoscale magnetic resonance measurements. Their physical properties predict them to have particularly useful applications in intracellular analysis. Before using them in intracellular experiments however, it should be clear whether diamond particles influence cell biology. While cytotoxicity has already been ruled out in previous studies, we consider the non-fatal influence of fluorescent nanodiamonds on the formation of reactive oxygen species (an important stress indicator and potential target for intracellular sensing) for the first time. We investigated the influence of different sizes, shapes and concentrations of nanodiamonds on the genetic and protein level involved in oxidative stress-related pathways of the HeLa cell, an important model cell line in research. The changes in viability of the cells and the difference in intracellular levels of free radicals, after diamond uptake, are surprisingly small. At lower diamond concentrations, the cellular metabolism cannot be distinguished from that of untreated cells. This research supports the claims of non-toxicity and includes less obvious non-fatal responses. Finally, we give a handhold concerning the diamond concentration and size to use for non-toxic, intracellular measurements in favour of (cancer) research in HeLa cells

The interaction of fluorescent nanodiamond probes with cellular media

Fluorescent nanodiamonds (FNDs) are promising tools to image cells, bioanalytes and physical quantities such as temperature, pressure, and electric or magnetic fields with nanometer resolution. To exploit their potential for intracellular applications, the FNDs have to be brought into contact with cell culture media. The interactions between the medium and the diamonds crucially influence sensitivity as well as the ability to enter cells. The authors demonstrate that certain proteins and salts spontaneously adhere to the FNDs and may cause aggregation. This is a first investigation on the fundamental questions on how (a) FNDs interact with the medium, and (b) which proteins and salts are being attracted. A differentiation between strongly binding and weakly binding proteins is made. Not all proteins participate in the formation of FND aggregates. Surprisingly, some main components in the medium seem to play no role in aggregation. Simple strategies to prevent aggregation are discussed. These include adding the proteins, which are naturally present in the cell culture to the diamonds first and then inserting them in the full medium. Graphical abstractSchematic of the interaction of nanodiamonds with cell culture medium. Certain proteins and salts adhere to the diamond surface and lead to aggregation or to formation of a protein corona

Circulating adrenomedullin estimates survival and reversibility of organ failure in sepsis: the prospective observational multinational Adrenomedullin and Outcome in Sepsis and Septic Shock-1 (AdrenOSS-1) study

Background: Adrenomedullin (ADM) regulates vascular tone and endothelial permeability during sepsis. Levels of circulating biologically active ADM (bio-ADM) show an inverse relationship with blood pressure and a direct relationship with vasopressor requirement. In the present prospective observational multinational Adrenomedullin and Outcome in Sepsis and Septic Shock 1 (, AdrenOSS-1) study, we assessed relationships between circulating bio-ADM during the initial intensive care unit (ICU) stay and short-term outcome in order to eventually design a biomarker-guided randomized controlled trial. Methods: AdrenOSS-1 was a prospective observational multinational study. The primary outcome was 28-day mortality. Secondary outcomes included organ failure as defined by Sequential Organ Failure Assessment (SOFA) score, organ support with focus on vasopressor/inotropic use, and need for renal replacement therapy. AdrenOSS-1 included 583 patients admitted to the ICU with sepsis or septic shock. Results: Circulating bio-ADM levels were measured upon admission and at day 2. Median bio-ADM concentration upon admission was 80.5 pg/ml [IQR 41.5-148.1 pg/ml]. Initial SOFA score was 7 [IQR 5-10], and 28-day mortality was 22%. We found marked associations between bio-ADM upon admission and 28-day mortality (unadjusted standardized HR 2.3 [CI 1.9-2.9]; adjusted HR 1.6 [CI 1.1-2.5]) and between bio-ADM levels and SOFA score (p < 0.0001). Need of vasopressor/inotrope, renal replacement therapy, and positive fluid balance were more prevalent in patients with a bio-ADM > 70 pg/ml upon admission than in those with bio-ADM ≤ 70 pg/ml. In patients with bio-ADM > 70 pg/ml upon admission, decrease in bio-ADM below 70 pg/ml at day 2 was associated with recovery of organ function at day 7 and better 28-day outcome (9.5% mortality). By contrast, persistently elevated bio-ADM at day 2 was associated with prolonged organ dysfunction and high 28-day mortality (38.1% mortality, HR 4.9, 95% CI 2.5-9.8). Conclusions: AdrenOSS-1 shows that early levels and rapid changes in bio-ADM estimate short-term outcome in sepsis and septic shock. These data are the backbone of the design of the biomarker-guided AdrenOSS-2 trial. Trial registration: ClinicalTrials.gov, NCT02393781. Registered on March 19, 2015

Hot Spot or Not: A Comparison of Spatial Statistical Methods to Predict Prospective Malaria Infections.

Within affected communities, Plasmodium falciparum infections may be skewed in distribution such that single or small clusters of households consistently harbour a disproportionate number of infected individuals throughout the year. Identifying these hotspots of malaria transmission would permit targeting of interventions and a more rapid reduction in malaria burden across the whole community. This study set out to compare different statistical methods of hotspot detection (SaTScan, kernel smoothing, weighted local prevalence) using different indicators (PCR positivity, AMA-1 and MSP-1 antibodies) for prediction of infection the following year. Two full surveys of four villages in Mwanza, Tanzania were completed over consecutive years, 2010-2011. In both surveys, infection was assessed using nested polymerase chain reaction (nPCR). In addition in 2010, serologic markers (AMA-1 and MSP-119 antibodies) of exposure were assessed. Baseline clustering of infection and serological markers were assessed using three geospatial methods: spatial scan statistics, kernel analysis and weighted local prevalence analysis. Methods were compared in their ability to predict infection in the second year of the study using random effects logistic regression models, and comparisons of the area under the receiver operating curve (AUC) for each model. Sensitivity analysis was conducted to explore the effect of varying radius size for the kernel and weighted local prevalence methods and maximum population size for the spatial scan statistic. Guided by AUC values, the kernel method and spatial scan statistics appeared to be more predictive of infection in the following year. Hotspots of PCR-detected infection and seropositivity to AMA-1 were predictive of subsequent infection. For the kernel method, a 1 km window was optimal. Similarly, allowing hotspots to contain up to 50% of the population was a better predictor of infection in the second year using spatial scan statistics than smaller maximum population sizes. Clusters of AMA-1 seroprevalence or parasite prevalence that are predictive of infection a year later can be identified using geospatial models. Kernel smoothing using a 1 km window and spatial scan statistics both provided accurate prediction of future infection

Substrate Specifity Profiling of the Aspergillus fumigatus Proteolytic Secretome Reveals Consensus Motifs with Predominance of Ile/Leu and Phe/Tyr

The filamentous fungus Aspergillus fumigatus (AF) can cause devastating infections in immunocompromised individuals. Early diagnosis improves patient outcomes but remains challenging because of the limitations of current methods. To augment the clinician's toolkit for rapid diagnosis of AF infections, we are investigating AF secreted proteases as novel diagnostic targets. The AF genome encodes up to 100 secreted proteases, but fewer than 15 of these enzymes have been characterized thus far. Given the large number of proteases in the genome, studies focused on individual enzymes may overlook potential diagnostic biomarkers.As an alternative, we employed a combinatorial library of internally quenched fluorogenic probes (IQFPs) to profile the global proteolytic secretome of an AF clinical isolate in vitro. Comparative protease activity profiling revealed 212 substrate sequences that were cleaved by AF secreted proteases but not by normal human serum. A central finding was that isoleucine, leucine, phenylalanine, and tyrosine predominated at each of the three variable positions of the library (44.1%, 59.1%, and 57.0%, respectively) among substrate sequences cleaved by AF secreted proteases. In contrast, fewer than 10% of the residues at each position of cleaved sequences were cationic or anionic. Consensus substrate motifs were cleaved by thermostable serine proteases that retained activity up to 50°C. Precise proteolytic cleavage sites were reliably determined by a simple, rapid mass spectrometry-based method, revealing predominantly non-prime side specificity. A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints. However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures.This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus. Expansion of this technique to protease secretion during infection could lead to development of novel approaches to fungal diagnosis

A fluorescent nanodiamond foundation for quantum sensing in cells

Free radicals play a major role in the aging process as well as a most diseases. However, we barely know anything about them. These tiny molecules have an extremely short lifespan and are difficult to measure, while their role in health related processes is considerable. Fluorescent nanodiamonds are very small diamonds which can shed a light on this research question. These diamonds emit a constant light in a controlled setting. This is possible due to a small defect in the structure of the diamond, which makes it fluorescent. When free radicals are present, the light changes, which allows measurement of the radicals. During my PhD I have laid the basis for these biological measurements. Not all cells automatically take up diamonds or the diamonds tend to aggregate in cellular medium. By changing the solutions in which we administer the diamonds, we can prevent these obstacles. I have also performed a very detailed analysis of the cellular response on diamond uptake. Conveniently, the cells hardly show any response to the diamond uptake, an important result for our future measurements. In addition, I have developed new ways of targeting the diamonds to specific places in the cells, to obtain location specific information. Finally I have determined the subcellular location of the diamonds using a new technique, based on integrated electron microscopy. During my work I have laid the foundation for promising cellular research of ageing and disease using fluorescent nanodiamonds

Nanodiamond uptake in colon cancer cells: the influence of direction and trypsin-EDTA treatment

Nanoparticles are routinely used in cell biology. They deliver drugs or function as labels or sensors. For many of these applications it is essential that the nanoparticles enter the cells. While some cell types readily ingest all kinds of particles, others just don't. We report that uptake can be enhanced for some cells if the particles are administered from the basolateral side of the cells (in this case from below). Compared to apical uptake (from above), we report an 8-fold increase in the number of fluorescent nanodiamonds internalized by the colon cancer cell line HT29. Up to 96% of the cells treated by a modified protocol contain at least one nanodiamond, whereas in the control group we could observe nanodiamonds in less than half of the cells. We were also able to show that simple treatment of cell clusters with trypsin-EDTA leads to the same enhancement of the nanodiamond uptake as seeding the cells on top of the nanoparticles. Although our study is focused on nanodiamonds in HT29 cells, we believe that this method could also be applicable for other nanoparticles and cells with a specific directionality