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We describe the process of generating a fluorophore-induced plasmonic current (FIPC) from copper nanoparticle films. Previous work and the literature have shown that excited near-field fluorophores are able to plasmonically couple with metal nanoparticle films (MNFs), inducing surface plasmons in the films. These induced surface plasmons are then in turn able to generate a directly measurable electrical current across the film. These generated currents have been quantified and detected in noble metal films, such as those made from Ag and Au, but due to the cost of such films, there has been a push to use lower cost materials for FIPC. Previous work has detailed the use of gold, silver, and aluminum films for these purposes, and in this paper, we will subsequently examine the ability of thermally deposited copper films to generate FIPC when in close proximity to excited near-field fluorophores. We report the effects of copper film thickness, the effects of light polarization and solution conductance, and the effects of metal-enhanced fluorescence (MEF) emission on the generation of plasmonic current.
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In this paper we demonstrate fluorophore induced plasmonic current (FIPC) from aluminum nanoparticle films. It has been previously shown that near-field excited fluorophores are able to plasmonically couple with metal nanoparticle films (MNF's) and induce surface plasmons, which in turn leads to a direct measurable electrical current through the MNF. These currents have been detected and quantified in noble metal MNF's, however due to future envisioned cost considerations there has been a push to adapt FIPC for use with less expensive metals. Subsequently, we observe that plasmonic aluminum films are able to produce these current changes when in close proximity to excited fluorophores, and the magnitude of the current changes are respective to the magnitude of the extinction coefficients of the fluorophores themselves. These findings also further support recent literature reports showing the inverse relationship between metal-enhanced fluorescence (MEF) and FIPC.
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Sample preparation is an essential step for nearly every type of biochemical analysis in use today. Among the most important of these analyses is the diagnosis of diseases, since their treatment may rely greatly on time and, in the case of infectious diseases, containing their spread within a population to prevent outbreaks. To address this, many different methods have been developed for use in the wide variety of settings for which they are needed. In this work, we have reviewed the literature and report on a broad range of methods that have been developed in recent years and their applications to point-of-care (POC), high-throughput screening, and low-resource and traditional clinical settings for diagnosis, including some of those that were developed in response to the coronavirus disease 2019 (COVID-19) pandemic. In addition to covering alternative approaches and improvements to traditional sample preparation techniques such as extractions and separations, techniques that have been developed with focuses on integration with smart devices, laboratory automation, and biosensors are also discussed.
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Ensaios de Triagem em Larga Escala/métodos , Manejo de Espécimes/métodos , Técnicas Biossensoriais/métodos , COVID-19 , Doenças Transmissíveis/diagnóstico , Ensaios de Triagem em Larga Escala/tendências , Humanos , Pandemias/prevenção & controle , Sistemas Automatizados de Assistência Junto ao Leito/tendências , Testes Imediatos/tendências , SARS-CoV-2RESUMO
While the utility of reactive oxygen species in photodynamic therapies for both cancer treatments and antimicrobial applications has received much attention, the inherent potential of reactive nitrogen species (RNS) including nitric oxide (NOË) for these applications should not be overlooked. In recent years, NOË donor species with numerous-including photodynamic-mechanisms have been classified with efficacy in antimicrobial and therapeutic applications. While properties of NOË delivery may be tuned structurally, herein we describe for the first time a method by which photodynamic NOË release is amplified simply by utilizing a plasmonic metal substrate. This is a process we term "metal-enhanced nitric oxide release", or ME-NOË. Using donor agents known as brominated carbon nanodots (BrCND), also the first carbon nanodot variation classified to release NOË photodynamically, and the fluorescence-on probe DAF-FM, we report metal-enhanced release of NOË 2- to 6-fold higher than what is achieved under classical conditions. Factors affecting the plasmon-amplified photodynamic system are subsequently studied, including exposure times, excitation powers, and surface area, and consistent ME-NOË factors are reported from BrCND across these tunable conditions. Only probe concentration is determined to impact the detected ME-NOË factor, with higher concentrations resulting in improved detectability of "actual" NOË release enhancement. Further, principles of metal-enhanced fluorescence (MEF) are applied to achieve a faster, high-throughput experimental method with improved data resolution in ME-NOË detection. The results have significant implications for the improvement of not just carbon nanodot NOË donor agents, but a wide spectrum of photoactivated NOË donor systems as well.
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Anti-Infecciosos , Fotoquimioterapia , Carbono , Fluorescência , Óxido NítricoRESUMO
Sample preparation is one of the most time-consuming steps in diagnostic assays, particularly those involving biological samples. In this paper we report the results of finite-difference time-domain (FDTD) simulations and thermographic imaging experiments carried out with the intent of designing a microplate for rapid, high-throughput sample preparation to aid diagnostic assays. This work is based on devices known as microwave lysing triangles (MLTs) that have been proven capable of rapid sample preparation when irradiated in a standard microwave cavity. FDTD software was used to model a microplate platform as a polystyrene substrate with an array of various passive scattering elements (PSEs) of different sizes, shapes, and interelement spacings in a 2.45 GHz field identical to that of a common microwave oven. Based on the FDTD modeling, several PSE arrays were fabricated by cutting PSEs out of aluminum foil and adhering them to the bottom of regular polystyrene microplates to make prototypes. Each prototype microplate was then irradiated in a microwave cavity for a range of different times, powers, and source angles and the heating effects were observed via a forward looking infrared (FLIR) camera. Based on the results, two prototype microplate platforms have been shown to demonstrate electromagnetic and thermal enhancements similar to those seen with MLTs as well as tunable thermal responses to radio frequency (RF) irradiation.
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The evolving threat of antibiotic resistance development in pathogenic bacteria necessitates the continued cultivation of new technologies and agents to mitigate associated negative health impacts globally. It is no surprise that infection prevention and control are cited by the Centers for Disease Control and Prevention (CDC) as two routes for combating this dangerous trend. One technology that has gained great research interest is antimicrobial photodynamic inactivation of bacteria, or APDI. This technique permits controllable activation of antimicrobial effects by combining specific light excitation with the photodynamic properties of a photosensitizer; when activated, the photosensitizer generates reactive oxygen species (ROS) from molecular oxygen via either a type I (electron transfer) or type II (energy transfer) pathway. These species subsequently inflict oxidative damage on nearby bacteria, resulting in suppressed growth and cell death. To date, small molecule photosensitizers have been developed, yet the scalability of these as widespread sterilization agents is limited due to complex and costly synthetic procedures. Herein we report the use of brominated carbon nanodots (BrCND) as new photosensitizers for APDI. These combustion byproducts are easily and inexpensively collected; incorporation of bromine into the nanodot permits photosensitization effects that are not inherent to the carbon nanodot structure alone-a consequence of triplet character gained by the heavy atom effect. BrCND demonstrate both type I and type II photosensitization under UV-A irradiation, and furthermore are shown to have significant antimicrobial effects against both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus and Listeria monocytogenes as well. A mechanism of "dark" toxicity is additionally reported; the pH-triggered release of reactive nitrogen species is detected from a carbon nanodot structure for the first time. The results described present the BrCND structure as a competitive new antimicrobial agent for controllable sterilization of bacteria.
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Anti-Infecciosos , Fotoquimioterapia , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Bactérias , Carbono , Fármacos Fotossensibilizantes/farmacologiaRESUMO
Antibiotic resistance development in bacteria is an ever-increasing global health concern as new resistant strains and/or resistance mechanisms emerge each day, out-pacing the discovery of novel antibiotics. Increasingly, research focuses on alternate techniques, such as antimicrobial photodynamic therapy (APDT) or photocatalytic disinfection, to combat pathogens even before infection occurs. Small molecule "photosensitizers" have been developed to date for this application, using light energy to inflict damage and death on nearby pathogens via the generation of reactive oxygen species (ROS). These molecular agents are frequently limited in widespread application by synthetic expense and complexity. Carbon dots, or fluorescent, quasi-spherical nanoparticle structures, provide an inexpensive and "green" solution for a new class of APDT photosensitizers. To date, reviews have examined the overall antimicrobial properties of carbon dot structures. Herein we provide a focused review on the recent progress for carbon nanodots in photodynamic disinfection, highlighting select studies of carbon dots as intrinsic photosensitizers, structural tuning strategies for optimization, and their use in hybrid disinfection systems and materials. Limitations and challenges are also discussed, and contemporary experimental strategies presented. This review provides a focused foundation for which APDT using carbon dots may be expanded in future research, ultimately on a global scale.
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In this work we investigate the relationship between metal-enhanced fluorescence (MEF) and fluorophore-induced plasmonic current (PC). This is accomplished through measurements of both radiative emission (MEF) and direct electrical current generation between discrete metal nanoparticles upon fluorophore excitation (PC). We have conducted these measurements on silver and gold nanoparticle island films, over a range of nanoparticle sizes and spacing in the films. We have observed an inverse relationship in the magnitude of MEF with PC, where larger and more closely spaced metal nanoparticles are found to result in increased PC and subsequently a decreased MEF. We attribute this effect to the relatively high capacitance and low charging energy of large and closely spaced particles, providing an outlet for plasmon relaxation in the form of electron flow and electrical current generation. These results are significant as they open potential for controlling for and the optimization of both MEF and PC.
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In this work, we report the surface-based electrical detection of singlet oxygen using the emerging fluorophore-induced plasmonic current (PC) technique. By this method, we utilize the fluorescent "turn on" response of the well-known singlet oxygen sensor green (SOSG) singlet oxygen (1O2) fluorescent probe for the generation of fluorophore-induced PC in a silver nanoparticle film. To demonstrate the potential utility of this new technique, a photosensitizing molecule is used to generate 1O2 in a solution containing the SOSG probe. The resulting change in SOSG fluorescence quantum yield and extinction coefficient permits stronger energy transfer from the SOSG probe to a proximal silver nanoparticle island film located in the near-electric field of the probe. This yields an increase in the induced electric current flow, allowing for the detection of the 1O2 analyte. To the author's knowledge, this represents the first detection of the reactive oxygen species 1O2 utilizing fluorophore-induced PC methodology and even broader electrical detection of 1O2. This is significant as it opens the possibility for 1O2 detection methods which do not require a traditional "photodetector" and associated optics, simplifying the instrumentation over existing fluorescence detection methods and potentially even lowering the cost.
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Oxigênio Singlete/química , Ressonância de Plasmônio de Superfície/métodosRESUMO
Metal-enhanced fluorescence (MEF) is a promising technology with impact in diagnostics, electronics, and sensing. Despite investigation into MEF fundamentals, some properties remain unresearched, notably spectral distortion. To date, publications have described its underpinnings, yet comprehensive analysis is needed, as presented recently for silver films. Herein we expand this description using zinc substrates (ZnNPs). Significant red-edge and blue-edge distortions are reported using Rose Bengal. Radiative decay rate modification is identified as key in amplifying fast/slow electronic transitions by the enhanced emission mechanism. Furthermore, we identify distortion in published studies, bolstering our thinking that spectral distortion is an intrinsic property of MEF.
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Metal-enhanced fluorescence (MEF) has become an increasingly important technology in recent years, with thorough research addressing the fundamentals of MEF. In many studies, spectral distortion is observed in the enhanced spectra as compared to free-space fluorescence emission profiles. Despite this observation, very little experimentation has hitherto been undertaken to investigate the mechanistic underpinnings of spectral distortion in MEF. Herein we investigate MEF spectral distortion using Rose Bengal and Fluorescein on silver nanoparticle substrates, subsequently isolating the coupled fluorescence spectrum for a deeper understanding of the spectral modifications. Clear experimental evidence for bathochromic distortion is reported. Remarkably, we also report hypsochromic distortion in one of the first experimental observations of plasmonic coupling to high-energy excited states. Additionally, the coupled fluorescence spectra from other published literature has also been both extracted and examined, and the subsequent spectral distortion reported here. The previously asserted theory of radiative decay rate modification for spectral distortion is discussed in the context of both plasmonic properties as well as fluorophore photophysical characteristics including lifetime and quantum yield. The dual enhancement mechanism of MEF is also explored in the context of spectral distortion. The results and discussion reported herein subsequently provide one of the first comprehensive examinations of spectral distortion in MEF to date.
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Proteolytic enzymes, which serve to degrade proteins to their amino acid building blocks, provide a distinct challenge for both diagnostics and biological research fields. Due to their ubiquitous presence in a wide variety of organisms and their involvement in disease, proteases have been identified as biomarkers for various conditions. Additionally, low-levels of proteases may interfere with biological investigation, as contamination with these enzymes can physically alter the protein of interest to researchers, resulting in protein concentration loss or subtler polypeptide clipping that leads to a loss of functionality. Low levels of proteolytic degradation also reduce the shelf-life of commercially important proteins. Many detection platforms have been developed to achieve low-concentration or low-activity detection of proteases, yet many suffer from limitations in analysis time, label stability, and ultimately sensitivity. Herein we demonstrate the potential utility of fluorescein derivatives as fluorescent labels in a new, turn-off enzymatic assay based on the principles of metal-enhanced fluorescence (MEF). For fluorescein sodium salt alone on nano-slivered 96-well plates, or Quanta Plates™, we report up to 11,000x enhancement for fluorophores within the effective coupling or enhancement volume region, defined as ~100â¯nm from the silver surface. We also report a 9% coefficient of variation, and detection on the picomolar concentration scale. Further, we demonstrate the use of fluorescein isothiocyanate-labeled YebF protein as a coating layer for a MEF-based, Quanta Plate™ enzymatic activity assay using trypsin as the model enzyme. From this MEF assay we achieve a detection limit of ~1.89â¯ng of enzyme (2.8 mBAEE activity units) which corresponds to a minimum fluorescence signal decrease of 10%. The relative success of this MEF assay sets the foundation for further development and the tuning of MEF platforms for proteolytic enzyme sensing not just for trypsin, but other proteases as well. In addition, we discuss the future development of ultra-fast detection of proteases via microwave-accelerated MEF (MAMEF) detection technologies.
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Ensaios Enzimáticos/métodos , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Tripsina/análise , Animais , Ensaios Enzimáticos/economia , Escherichia coli/química , Proteínas de Escherichia coli/química , Humanos , Proteólise , Espectrometria de Fluorescência/economia , Espectrometria de Fluorescência/métodos , Fatores de TempoRESUMO
Rapid sample preparation is one of the leading bottlenecks to low-cost and efficient sample component detection. To overcome this setback, a technology known as Lyse-It has been developed to rapidly (less than 60 seconds) lyse Gram-positive and-negative bacteria alike, while simultaneously fragmenting DNA/RNA and proteins into tunable sizes. This technology has been used with a variety of organisms, but the underlying mechanism behind how the technology actually works to fragment DNA/RNA and proteins has hitherto been studied. It is generally understood how temperature affects cellular lysing, but for DNA/RNA and protein degradation, the temperature and amount of energy introduced by microwave irradiation of the sample, cannot explain the degradation of the biomolecules to the extent that was being observed. Thus, an investigation into the microwave generation of reactive oxygen species, in particular singlet oxygen, hydroxyl radicals, and superoxide anion radicals, was undertaken. Herein, we probe one aspect, the generation of reactive oxygen species (ROS), which is thought to contribute to a non-thermal mechanism behind biomolecule fragmentation with the Lyse-It technology. By utilizing off/on (Photoinduced electron transfer) PET fluorescent-based probes highly specific for reactive oxygen species, it was found that as oxygen concentration in the sample and/or microwave irradiation power increases, more reactive oxygen species are generated and ultimately, more oxidation and biomolecule fragmentation occurs within the microwave cavity.
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Métodos Analíticos de Preparação de Amostras/métodos , Técnicas Bacteriológicas/métodos , Fragmentação do DNA/efeitos dos fármacos , Detergentes/farmacologia , Estabilidade de RNA/efeitos dos fármacos , Fragmentação do DNA/efeitos da radiação , DNA Bacteriano/química , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/efeitos da radiação , Hidrólise/efeitos da radiação , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/genética , Listeria monocytogenes/efeitos da radiação , Micro-Ondas , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , Oxigênio/análise , Oxigênio/metabolismo , Proteólise/efeitos dos fármacos , Proteólise/efeitos da radiação , Estabilidade de RNA/efeitos da radiação , RNA Bacteriano/química , RNA Bacteriano/efeitos dos fármacos , RNA Bacteriano/efeitos da radiação , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/efeitos da radiação , Temperatura , Fatores de Tempo , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/genética , Vibrio cholerae/efeitos da radiaçãoRESUMO
Nucleases are enzymes that can degrade genomic DNA and RNA that decrease the accuracy of quantitative measures of those nucleic acids. Here, we study conventional heating, standard microwave irradiation, and Lyse-It, a microwave-based lysing technology, for the potential to fragment and inactivate DNA and RNA endonucleases. Lyse-It employs the use of highly focused microwave irradiation to the sample ultimately fragmenting and inactivating RNase A, RNase B, and DNase I. Nuclease size and fragmentation were determined visually and quantitatively by SDS polyacrylamide gel electrophoresis and the mini-gel Agilent 2100 Bioanalyzer system, with a weighted size calculated to depict the wide range of nuclease fragmentation. Enzyme activity assays were conducted, and the rates were calculated to determine the effect of various lysing conditions on each of the nucleases. The results shown in this paper clearly demonstrate that Lyse-It is a rapid and highly efficient way to degrade and inactivate nucleases so that nucleic acids can be retained for down-stream detection.
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Desoxirribonuclease I/química , Fragmentos de Peptídeos/análise , Ribonuclease Pancreático/química , Ribonucleases/química , DNA/química , Desoxirribonuclease I/efeitos dos fármacos , Desoxirribonuclease I/efeitos da radiação , Detergentes/farmacologia , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Hidrólise , Micro-Ondas , Peso Molecular , Proteólise/efeitos dos fármacos , Proteólise/efeitos da radiação , RNA/química , Ribonuclease Pancreático/efeitos dos fármacos , Ribonuclease Pancreático/efeitos da radiação , Ribonucleases/efeitos dos fármacos , Ribonucleases/efeitos da radiação , SoluçõesRESUMO
The ability for safe and rapid pathogenic sample transportation and subsequent detection is an increasing challenge throughout the world. Herein, we describe and use bead-beating, vortex, sonication, 903 protein saver cards, and Lyse-It methods, aiming to inactivate Gram-positive and -negative bacteria with subsequent genome DNA (quantitative Polymerase Chain Reaction) qPCR detection. The basic concepts behind the four chosen technologies is their versatility, cost, and ease of use in developed and underdeveloped countries. The four methods target the testing of bacterial resilience, cellular extraction from general and complex media and subsequent DNA extraction for qPCR detection and amplification. These results demonstrate that conventional high temperature heating, 903 protein saver cards, and Lyse-It are all viable options for inactivating bacterial growth for safe shipping. Additionally, Lyse-It was found to be particularly useful as this technology can inactivate bacteria, extract cells from 903 protein saver cards, lyse bacterial cells, and additionally keep genomic DNA viable for qPCR detection.
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Fracionamento Celular/métodos , DNA Bacteriano/normas , Técnicas de Diagnóstico Molecular/métodos , Fracionamento Celular/economia , Fracionamento Celular/normas , DNA Bacteriano/química , Bactérias Gram-Negativas/química , Bactérias Gram-Positivas/química , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Kit de Reagentes para Diagnóstico/normasRESUMO
Many diagnostic fluorescence assays are limited by sensitivity (signal/noise) and minimum sample volume requirements. Herein we report a new, silvered conical-bottom 96-well plate platform used to increase the detectability from very small volumes of micromolar concentrations of fluorophores. This technology employs the principles of metal-enhanced fluorescence (MEF), which is the process by which fluorescence emission is amplified in the near-field of plasmonic nanoparticles. By combining the MEF effect with the advantages of a small volume conical well, we report and characterize detectable emission from fluorescent solutions down to 3 microliters in volume. We report enhancement factors for fluorescein and Rhodamine 6G and correlate these factors to the synchronous scattering spectra of the silvered conical wells. Subsequently, we determine corrected enhancement factors and discuss enhancement in terms of the MEF volume ratio effect and per mole of enhanced fluorophore. The research reported herein sets the foundation for future development of even more powerful MEF-based diagnostic assays.
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Brominated carbon nanodots are a new carbon nanostructure that exhibits strong phosphorescence without fixation. Herein we report plasmonic amplification of this phosphorescence in silver-coated Quanta Plate™ wells, a technique called metal-enhanced phosphorescence (MEP). Subsequently we correlate the excitation and emission components of brominated carbon nanodots to their respective enhancement values. These properties are then discussed in relation to the synchronous scattering spectrum of the plasmonic substrate, in the first report of its kind for MEP. These results set the foundation for expanded application of carbon nanodots, as the photophysical characteristics of phosphorescence are improved, and augment the growing understanding of MEP.
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Sample preparation is a leading bottleneck in rapid detection of pathogenic bacteria. Here, we use Lyse-It® for bacterial cellular lysis, genomic DNA fragmentation, and protein release and degradation for both Listeria monocytogenes and Vibrio cholerae. The concept of Lyse-It® employs a conventional microwave and Lyse-It® slides for intensely focused microwave irradiation onto the sample. High microwave power and a <60 second irradiation time allow for rapid cellular lysis and subsequent intracellular component release. The pathogenic bacteria are identified by quantitative polymerase chain reaction (qPCR), which subsequently demonstrates the viability of DNA for amplification post microwave-induced lysis. Intracellular component release, degradation, and detection of L. monocytogenes and V. cholerae has been performed and shown in this paper. These results demonstrate a rapid, low-cost, and efficient way for bacterial sample preparation on both food and water-borne Gram-positive and -negative organisms alike.
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Técnicas Bacteriológicas , Listeria monocytogenes , Vibrio cholerae , Animais , DNA Bacteriano , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Micro-Ondas , Reação em Cadeia da Polimerase , Ovinos , Temperatura , Vibrio cholerae/genética , Vibrio cholerae/isolamento & purificaçãoRESUMO
Carbon nanodots are nanometer sized fluorescent particles studied for their distinct photoluminescent properties and biocompatibility. Although extensive literature reports the modification and application of carbon nanodot fluorescence, little has been published pertaining to phosphorescence emission from carbon nanodots. The use of phosphors in biological imaging can lead to clearer detection, as the long lifetimes of phosphorescent emission permit off-gated collection that avoids noise from biological autofluorescence. Carbon nanodots present a desirable scaffold for this application, with advantageous qualities ranging from photostability to multi-color emission. This research reports the generation of a novel phosphorescent "heavy carbon" nanodot via halogenation of the carbon nanodot structure. By employing a collection pathway that effectively incorporates bromine into the nanostructure, T1 triplet character is introduced, and subsequently phosphorescence is observed in liquid media at room temperature for the first time in the nanodot literature. Further experiments are reported characterizing the conditions of observed phosphorescence and its pH-dependence. Our approach for producing "heavy carbon nanodots" is a low-cost and relatively simple method for generating the phosphorescent nanodots, which sets the foundation for its potential future use as a phosphorescent probe in application.