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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.

Explains the principles and current thinking behind plasmon enhanced Fluorescence Describes the current developments in Surface Plasmon Enhanced, Coupled and Controlled Fluorescence Details methods used to understand solar energy conversion, detect and quantify DNA more quickly and accurately, and enhance the timeliness and accuracy of digital.

Significance Coral reefs are highly productive ecosystems that raise a conundrum called “Darwin’s paradox”: How can high production flourish in low-nutrient conditions? We show here that in three abundant Caribbean sponges, the granules that have been commonly observed in sponge tissue for decades are polyphosphate granules. These granules can account for up to 40% of the total phosphorus (P) in sponge tissue. This finding has important implications for understanding P sequestration and recycling in the reef environment. We provide evidence that these granules are of bacterial origin and propose a P sequestration pathway by microbial symbionts and the sponge hosts. Considering the ancient origin of both partners, this process may have had an impact on the P cycle in Earth’s early history. 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.

We report on the recent advancements in the sensing of proteins, both directly and with the use of a fluorescent probe, through the use of Fluorophore-Induced Plasmonic Current (FIPC). FIPC are a phenomenon where a fluorophore or excited state species is in close proximity to a plasmonically active metal nanoparticle film (MNF), and the excited state is able to couple to the particle, ultimately leading to enhanced spectroscopic properties. This phenomenon is similar to the well-reported metal-enhanced fluorescence (MEF) phenomenon, wherein the coupled complex produces an enhanced fluorescence emission and a shorter lifetime. However, if the particles themselves are sufficiently spaced and oriented, an induced current can transfer from each discreet particle to the next, creating a detectable current across the film. This detectable current has a magnitude that is proportional to the fluorescent properties of the species that produced it, and can be affected by the polarization of the excitation source; the spacing and size of the particles on the film; the overlap between the spectral properties of the film and the species; as well as externally applied voltages and currents. In this study, we examined whether it is possible to detect protein species, directly due to both their intrinsic fluorescent and absorptive properties, and how that compares to commercially available protein detection probes, in a similar manner to prior work by our group addressing analyte detection via turn-on fluorescent probes. This FIPC-based detection technique is a novel method that has not been used for the detection of proteins, and the use of this method could expand the dynamic sensing range of first-pass testing, while overcoming some of the physical limitations on the upper limit of detection of both absorption spectroscopy and fluorescence emission spectroscopy. Our experiments sought to highlight the selectivity of FIPC-based detection relative to both fluorescence and absorption spectroscopy, as well as its sensitivity when working with protein analytes. We examined the effects of protein concentration, intrinsic fluorescent properties, and turn-on probes, as well as how these techniques compare to traditional analytical techniques used today.

We report on the detection and quantification of aqueous DNA by a fluorophore-induced plasmonic current (FIPC) sensing method. FIPC is a mechanism described by our group in the literature where a fluorophore in close proximity to a plasmonically active metal nanoparticle film (MNF) is able to couple with it, when in an excited state. This coupling produces enhanced fluorescent intensity from the fluorophore–MNF complex, and if conditions are met, a current is generated in the film that is intrinsically linked to the properties of the fluorophore in the complex. The magnitude of this induced current is related to the spectral properties of the film, the overlap between these film properties and those of the fluorophore, the spacing between the nanoparticles in the film, the excitation wavelength, and the polarization of the excitation source. Recent literature has shown that the FIPC system is ideal for aqueous ion sensing using turn-on fluorescent probes, and in this paper, we subsequently examine if it is possible to detect aqueous DNA also via a turn-on fluorescent probe, as well as other commercially available DNA detection strategies. We report the effects of DNA concentration, probe concentration, and probe characteristics on the development of an FIPC assay for the detection of non-specific DNA in aqueous solutions.

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.

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.

In this article, we report metal-enhanced singlet oxygen generation (ME¹O₂). We demonstrate a direct relationship between the singlet oxygen yield of a common photosensitizer (Rose Bengal) and the theoretical electric field enhancement or enhanced absorption of the photosensitizer in proximity to metallic nanoparticles. Using a series of photosensitizers, sandwiched between silver island films (SiFs), we report that the extent of singlet oxygen enhancement is inversely proportional to the free space singlet oxygen quantum yield. By modifying plasmon coupling parameters, such as nanoparticle size and shape, fluorophore/particle distance, and the excitation wavelength of the coupling photosensitizer, we can readily tune singlet oxygen yields for applications in singlet oxygen-based clinical therapy.

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.

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.