Sample Preparation and Diagnostic Methods for a Variety of Settings: A Comprehensive Review.

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.

Spectral Distortions in Zinc-based Metal-Enhanced Fluorescence Underpinned by Fast and Slow Electronic Transitions.

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.

Heavy carbon nanodots 2: plasmon amplification in Quanta Plate™ wells and the correlation with the synchronous scattering spectrum.

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.

Heavy carbon nanodots: a new phosphorescent carbon nanostructure.

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.

Phosphorus sequestration in the form of polyphosphate by microbial symbionts in marine sponges.

Marine sponges are major habitat-forming organisms in coastal benthic communities and have an ancient origin in evolution history. Here, we report significant accumulation of polyphosphate (polyP) granules in three common sponge species of the Caribbean coral reef. The identity of the polyP granules was confirmed by energy-dispersive spectroscopy (EDS) and by the fluorescence properties of the granules. Microscopy images revealed that a large proportion of microbial cells associated with sponge hosts contained intracellular polyP granules. Cyanobacterial symbionts cultured from sponges were shown to accumulate polyP. We also amplified polyphosphate kinase (ppk) genes from sponge DNA and confirmed that the gene was expressed. Based on these findings, we propose here a potentially important phosphorus (P) sequestration pathway through symbiotic microorganisms of marine sponges. Considering the widespread sponge population and abundant microbial cells associated with them, this pathway is likely to have a significant impact on the P cycle in benthic ecosystems.

Microwave-accelerated method for ultra-rapid extraction of Neisseria gonorrhoeae DNA for downstream detection.

Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by detection of the genomic target often involving polymerase chain reaction (PCR)-based approaches. In an effort to improve on current detection approaches, we have developed a unique two-step microwave-accelerated approach for rapid extraction and detection of Neisseria gonorrhoeae (gonorrhea, GC) DNA. Our approach is based on the use of highly focused microwave radiation to rapidly lyse bacterial cells, release, and subsequently fragment microbial DNA. The DNA target is then detected by a process known as microwave-accelerated metal-enhanced fluorescence (MAMEF), an ultra-sensitive direct DNA detection analytical technique. In the current study, we show that highly focused microwaves at 2.45 GHz, using 12.3-mm gold film equilateral triangles, are able to rapidly lyse both bacteria cells and fragment DNA in a time- and microwave power-dependent manner. Detection of the extracted DNA can be performed by MAMEF, without the need for DNA amplification, in less than 10 min total time or by other PCR-based approaches. Collectively, the use of a microwave-accelerated method for the release and detection of DNA represents a significant step forward toward the development of a point-of-care (POC) platform for detection of gonorrhea infections.

Examining outcome of early physician specialist assessment in injured workers with shoulder complaints.

BACKGROUND: There is minimal research on demographics, type of injury and diagnosis of injured workers with shoulder problems. The purposes of this study were: 1) to document the demographics of patients with shoulder complaints referred to an Early Shoulder Physician Assessment (ESPA) Program and to describe the recommended management, and 2) to examine the relationship between patient characteristics and their subjective complaints of pain and functional difficulty. METHODS: This study involved a retrospective review of electronic files of injured workers mostly seen within the first 16 weeks of injury or recurrence. Measures of functional difficulty and pain were the Quick Disabilities of the Arm, Shoulder and Hand (QuickDASH) and Numeric Pain Scale (NPS). RESULTS: Files of 550 consecutive patients, 260 females (47%), 290 men (53%) were examined. The average age was 49 (SD = 11, range 22-77), with 28 (5%) patients being 65 years of age or older. Patients who were not working were the most disabled group based on Quick DASH (F = 49.93, p < 0.0001) and NPS (F = 10.24, p = 0.002). Patients who were working full time performing regular duties were the least disabled according to both measures, the QuickDASH (F = 10.24, p = 0.002) and NPS (F = 7.57, p = 0.006). Patients waiting more than 16 weeks were slightly older (53 years of age vs. 49, p = 0.045) than those who met the criteria for early assessment with similar levels of pain and functional difficulty. Biceps pathology had the highest prevalence (37%). Full thickness tear had a prevalence of 14%. Instability, labral lesions and osteoarthritis of glenohumeral joint were uncommon conditions (3, 2 and 1% respectively). Fifty-five patients (10%) were surgical candidates and had higher scores on QuickDASH (F = 7.16, p = 0.008) and NPS (F = 4.24, p = 0.04) compared to those who did not require surgery. CONCLUSIONS: This study provides information on characteristics and prevalence of important variables in injured workers with shoulder problems and highlights the impact of these characteristics on pain and disability.

5-color multiplexed microwave-accelerated metal-enhanced fluorescence: detection and analysis of multiple DNA sequences from within one sample well within a few seconds.

We present a potentially highly sensitive and selective bio-assay for the potential detection of any five different DNA sequences from one sample in one well. The assay is based on a DNA "rapid catch and signal" (DNA-RCS) technology developed for the detection of different DNA sequences from a sample well area. Our signal amplification utilizes the metal-enhanced fluorescence (MEF) of dyes attached to the probe-DNAs, which hybridizes with the pre-formed mixture of anchor-DNA scaffolds on silver island films (SiFs). Low-power microwave irradiation accelerates both the formation of the anchor-DNA scaffold on the SiF-surface and anchor/probe DNA hybridization, i.e. "rapid catch" of target DNAs from a bulk solution, decreasing the assay run time from hours to only a few seconds. Localization of signaling dye-labels close to the SiFs make them extremely photostable, which allows for collecting/integrating the signal over a long time period. To demonstrate a 5 color DNA assay (5-plex) we have used a range of readily available Alexa™ dyes. Advantages and perspectives of the RCS-technologies ability to detect 5 different DNA sequences from within one plate-well are discussed.

Fluorescence-based Broad Dynamic Range Viscosity Probes.

We introduce two new fluorescent viscosity probes, SYBR Green (SG) and PicoGreen (PG), that we have studied over a broad range of viscosity and in collagen solutions. In water, both dyes have low quantum yields and excited state lifetimes, while in viscous solvents or in complex with DNA both parameters dramatically (300-1000-fold) increase. We show that in log-log scale the dependence of the dyes' quantum yield vs. viscosity is linear, the slope of which is sensitive to temperature. Application of SG and PG, as a fluorescence-based broad dynamic range viscosity probes, to the life sciences is discussed.

Fluorophore-Induced Plasmonic Current Generation from Copper Nanoparticle Films.

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.

Blind evaluation of the microwave-accelerated metal-enhanced fluorescence ultrarapid and sensitive Chlamydia trachomatis test by use of clinical samples.

Accurate point-of-care (POC) diagnostic tests for Chlamydia trachomatis infection are urgently needed for the rapid treatment of patients. In a blind comparative study, we evaluated microwave-accelerated metal-enhanced fluorescence (MAMEF) assays for ultrafast and sensitive detection of C. trachomatis DNA from vaginal swabs. The results of two distinct MAMEF assays were compared to those of nucleic acid amplification tests (NAATs). The first assay targeted the C. trachomatis 16S rRNA gene, and the second assay targeted the C. trachomatis cryptic plasmid. Using pure C. trachomatis, the MAMEF assays detected as few as 10 inclusion-forming units/ml of C. trachomatis in less than 9 min, including DNA extraction and detection. A total of 257 dry vaginal swabs from 245 female adolescents aged 14 to 22 years were analyzed. Swabs were eluted with water, the solutions were lysed to release and to fragment genomic DNA, and MAMEF-based DNA detection was performed. The prevalence of C. trachomatis by NAATs was 17.5%. Of the 45 samples that were C. trachomatis positive and the 212 samples that were C. trachomatis negative by NAATs, 33/45 and 197/212 were correctly identified by the MAMEF assays if both assays were required to be positive (sensitivity, 73.3%; specificity, 92.9%). Using the plasmid-based assay alone, 37/45 C. trachomatis-positive and 197/212 C. trachomatis-negative samples were detected (sensitivity, 82.2%; specificity, 92.9%). Using the 16S rRNA assay alone, 34/45 C. trachomatis-positive and 197/212 C. trachomatis-negative samples were detected (sensitivity, 75.5%; specificity, 92.9%). The overall rates of agreement with NAAT results for the individual 16S rRNA and cryptic plasmid assays were 89.5% and 91.0%, respectively. Given the sensitivity, specificity, and rapid detection of the plasmid-based assay, the plasmid-based MAMEF assay appears to be suited for clinical POC testing.

Highly sensitive quantitation of human serum albumin in clinical samples for hypoproteinemia using metal-enhanced fluorescence.

In this paper, we have explored metal-enhanced fluorescence (MEF) of the Human serum albumin indicators: Albumin Blue 580, Merocyanine 540 and Bromophenol Blue in close proximity to silver nano-particles, SiFs, from both buffered and clinical samples. The photostability of the Albumin Blue 580 is shown to be much more prolonged from the SiFs as compared to glass (a control sample), potentially allowing for longer detection times to further improve assay statistics. Our findings suggest the widespread use of nanoparticulate SiFs surfaces for the enhanced detection of HSA, particularly for Hypoproteinemia, where an enhanced assay performance at low protein abundance is required.

Metal-enhanced fluorescence based excitation volumetric effect of plasmon-enhanced singlet oxygen and super oxide generation.

In this contribution we show that the Metal-Enhanced Fluorescence (MEF) Excitation Volumetric Effect (EVE), has a profound effect on the formation of Reactive Oxygen Species (ROS), such as singlet oxygen ((1)O2) and superoxide anion radical (O2(-)*), when sensitizers are placed in close proximity to plasmon supporting nanoparticulate substrates. In particular, when the singlet oxygen sensitizer rose bengal is placed on a SiFs surface, i.e. on a silver island film, the (1)O2 response to power is non-linear, and at 100 mW excitation power (535 nm) it is about 5 times higher, as compared to glass control samples, measured with the commercially available (1)O2 probe Sensor Green™. We also report a similar power dependence of superoxide generation for acridine on SiFs surfaces, but using the dihydroethidium O2(-)* probe (DHE). Our findings are consistent with our previously postulated Metal-Enhanced Fluorescence (MEF) and EVE models.

Experimental and theoretical study of the distance dependence of metal-enhanced fluorescence, phosphorescence and delayed fluorescence in a single system.

Distance dependent singlet and triplet metal-enhanced emission of eosin from silica coated silver island films (SiFs) has been studied by steady-state and time resolved fluorescence techniques, along with theoretical finite difference time domain (FDTD) numerical simulations, to understand how the thickness of the dielectric coating surrounding silver nanoparticles fundamentally affects luminescence enhancement. Our findings suggest that the distance dependence of metal-enhanced phenomena such as fluorescence, phosphorescence and delayed fluorescence is underpinned by the decay of the electric near-field, and depending on the actual silver silica sample embodiment, one can see either decreased or enhanced luminescence. These results not only expand our current MEF thinking but also suggest that one may well be able to approximate plasmon-enhanced luminescence values.

Fluorophore-Induced Plasmonic Current Generation from Aluminum Nanoparticle Films.

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.

Ultra-fast pg/ml anthrax toxin (protective antigen) detection assay based on microwave-accelerated metal-enhanced fluorescence.

Rapid presymptomatic diagnosis of Bacillus anthracis at early stages of infection plays a crucial role in prompt medical intervention to prevent rapid disease progression and accumulation of lethal levels of toxin. To detect low levels of the anthrax protective antigen (PA) exotoxin in biological fluids, we have developed a metal-enhanced fluorescence (MEF)-PA assay using a combination of the MEF effect and microwave-accelerated PA protein surface absorption. The assay is based on a modified version of our "rapid catch and signal" (RCS) technology previously designed for the ultra-fast and sensitive analysis of genomic DNA sequences. Technologically, the proposed MEF-PA assay uses standard 96-well plastic plates modified with silver island films (SiFs) grown within the wells. It is shown that the fluorescent probe, covalently attached to the secondary antibody, plays a crucial role of indicating complex formation (i.e., shows a strong MEF response to the recognition event). Microwave irradiation rapidly accelerates PA deposition onto the surface ("rapid catch"), significantly speeding up the MEF-PA assay and resulting in a total assay run time of less than 40 min with an analytical sensitivity of less than 1 pg/ml PA.

Reduced lifetimes are directly correlated with excitation irradiance in metal-enhanced fluorescence (MEF).

We describe a fundamental observation in Metal-Enhanced Fluorescence (MEF), which has become a leading technology in the life sciences today, namely, how the lifetime of fluorophores near-to metallic plasmon-supporting silver islands/nanoparticles, modulates as a function of excitation power irradiance. This finding is in stark contrast to that observed in classical far-field fluorescence spectroscopy, where excitation power does not influence fluorophore radiative decay/lifetime.