Ultrasensitive Protein Detection Technologies for Extracellular Vesicle Measurements.

Extracellular vesicles (EVs) are nanoscopic, heterogenous, lipid-rich particles that carry a multitude of cargo biomolecules including proteins, nucleic acids, and metabolites. Although historically EVs were regarded as cellular debris with no intrinsic value, growing understanding of EV biogenesis has led to the realization that EVs facilitate intercellular communication and are sources of liquid biomarkers. EVs can be isolated and analyzed from a wide variety of accessible biofluids for biomarker discovery and diagnostic applications. There is a diversity of EVs from different biological compartments (e.g., cells and tissues), and some of these EVs are present at extremely low concentrations. Consequently, a challenge in the field is to find appropriate markers that enable selective isolation of these rare EVs. Many conventional protein detection technologies have limited sensitivity to detect low abundance biomarkers in EVs, limiting their use in EV research. Advances in ultrasensitive detection technologies are needed to harness the potential of EVs for clinical application. This Perspective highlights current EV research focusing on ultrasensitive detection technologies, their limitations, and areas of potential growth in the future.

Proteomics of prostate cancer serum and plasma using low and high throughput approaches.

Despite progress, MS-based proteomics in biofluids, especially blood, faces challenges such as dynamic range and throughput limitations in biomarker and disease studies. In this work, we used cutting-edge proteomics technologies to construct label-based and label-free workflows, capable of quantifying approximately 2,000 proteins in biofluids. With 70µL of blood and a single depletion strategy, we conducted an analysis of a homogenous cohort (n = 32), comparing medium-grade prostate cancer patients (Gleason score: 7(3 + 4); TNM stage: T2cN0M0, stage IIB) to healthy donors. The results revealed dozens of differentially expressed proteins in both plasma and serum. We identified the upregulation of Prostate Specific Antigen (PSA), a well-known biomarker for prostate cancer, in the serum of cancer cohort. Further bioinformatics analysis highlighted noteworthy proteins which appear to be differentially secreted into the bloodstream, making them good candidates for further exploration.

Frontiers in plasma proteome profiling platforms: innovations and applications.

Biomarkers play a crucial role in advancing precision medicine by enabling more targeted and individualized approaches to diagnosis and treatment. Various biofluids, including serum, plasma, cerebrospinal fluid (CSF), saliva, tears, pancreatic cyst fluids, and urine, have been identified as rich sources of potential for the early detection of disease biomarkers in conditions such as cancer, cardiovascular diseases, and neurodegenerative disorders. The analysis of plasma and serum in proteomics research encounters challenges due to their high complexity and the wide dynamic range of protein abundance. These factors impede the sensitivity, coverage, and precision of protein detection when employing mass spectrometry, a widely utilized technology in discovery proteomics. Conventional approaches such as Neat Plasma workflow are inefficient in accurately quantifying low-abundant proteins, including those associated with tissue leakage, immune response molecules, interleukins, cytokines, and interferons. Moreover, the manual nature of the workflow poses a significant hurdle in conducting large cohort studies. In this study, our focus is on comparing workflows for plasma proteomic profiling to establish a methodology that is not only sensitive and reproducible but also applicable for large cohort studies in biomarker discovery. Our investigation revealed that the Proteograph XT workflow outperforms other workflows in terms of plasma proteome depth, quantitative accuracy, and reproducibility while offering complete automation of sample preparation. Notably, Proteograph XT demonstrates versatility by applying it to various types of biofluids. Additionally, the proteins quantified widely cover secretory proteins in peripheral blood, and the pathway analysis enriched with relevant components such as interleukins, tissue necrosis factors, chemokines, and B and T cell receptors provides valuable insights. These proteins, often challenging to quantify in complex biological samples, hold potential as early detection markers for various diseases, thereby contributing to the improvement of patient care quality.

Recent advancements in non-invasive wearable electrochemical biosensors for biomarker analysis - A review.

A biomarker is a molecular indicator that can be used to identify the presence or severity of a disease. It may be produced due to biochemical or molecular changes in normal biological processes. In some cases, the presence of a biomarker itself is an indication of the disease, while in other cases, the elevated or depleted level of a particular protein or chemical substance aids in identifying a disease. Biomarkers indicate the progression of the disease in response to therapeutic interventions. Identifying these biomarkers can assist in diagnosing the disease early and providing proper therapeutic treatment. In recent years, wearable electrochemical (EC) biosensors have emerged as an important tool for early detection due to their excellent selectivity, low cost, ease of fabrication, and improved sensitivity. There are several challenges in developing a fully integrated wearable sensor, such as device miniaturization, high power consumption, incorporation of a power source, and maintaining the integrity and durability of the biomarker for long-term continuous monitoring. This review covers the recent advancements in the fabrication techniques involved in device development, the types of sensing platforms utilized, different materials used, challenges, and future developments in the field of wearable biosensors.

Heat Transfer by Sweat Droplet Evaporation.

Sweating regulates the body temperature in extreme environments or during exercise. Here, we investigate the evaporative heat transfer of a sweat droplet at the microscale to unveil how the evaporation complexity of a sweat droplet would affect the body's ability to cool under specific environmental conditions. Our findings reveal that, depending on the relative humidity and temperature levels, sweat droplets experience imperfect evaporation dynamics, whereas water droplets evaporate perfectly at equivalent ambient conditions. At low humidity, the sweat droplet fully evaporates and leaves a solid deposit, while at high humidity, the droplet never reaches a solid deposit and maintains a liquid phase residue for both low and high temperatures. This unprecedented evaporation mechanism of a sweat droplet is attributed to the intricate physicochemical properties of sweat as a biofluid. We suppose that the sweat residue deposited on the surface by evaporation is continuously absorbing the surrounding moisture. This route leads to reduced evaporative heat transfer, increased heat index, and potential impairment of the body's thermoregulation capacity. The insights into the evaporative heat transfer dynamics at the microscale would help us to improve the knowledge of the body's natural cooling mechanism with practical applications in healthcare, materials science, and sports science.

Advances in electrochemical biosensor design for the detection of the stress biomarker cortisol.

The monitoring of stress levels in humans has become increasingly relevant, given the recent incline of stress-related mental health disorders, lifestyle impacts, and chronic physiological diseases. Long-term exposure to stress can induce anxiety and depression, heart disease, and risky behaviors, such as drug and alcohol abuse. Biomarker molecules can be quantified in biological fluids to study human stress. Cortisol, specifically, is a hormone biomarker produced in the adrenal glands with biofluid concentrations that directly correlate to stress levels in humans. The rapid, real-time detection of cortisol is necessary for stress management and predicting the onset of psychological and physical ailments. Current methods, including mass spectrometry and immunoassays, are effective for sensitive cortisol quantification. However, these techniques provide only single measurements which pose challenges in the continuous monitoring of stress levels. Additionally, these analytical methods often require trained personnel to operate expensive instrumentation. Alternatively, low-cost electrochemical biosensors enable the real-time detection and continuous monitoring of cortisol levels while also providing adequate analytical figures of merit (e.g., sensitivity, selectivity, sensor response times, detection limits, and reproducibility) in a simple design platform. This review discusses the recent developments in electrochemical biosensor design for the detection of cortisol in human biofluids. Special emphasis is given to biosensor recognition elements, including antibodies, molecularly imprinted polymers (MIPs), and aptamers, as critical components of electrochemical biosensors for cortisol detection. Furthermore, the advantages and limiting factors of various electrochemical techniques and sensing in complex biofluid matrices are overviewed. Remarks on the current challenges and future perspectives regarding electrochemical biosensors for stress monitoring are provided, including matrix effects (pH dependence and biological interferences), wearability, and large-scale production.

Spectral effects and enhancement quantification in healthy human saliva with surface-enhanced Raman spectroscopy using silver nanopillar substrates.

OBJECTIVES: Raman spectroscopy as a diagnostic tool for biofluid applications is limited by low inelastic scattering contributions compared to the fluorescence background from biomolecules. Surface-enhanced Raman spectroscopy (SERS) can increase Raman scattering signals, thereby offering the potential to reduce imaging times. We aimed to evaluate the enhancement related to the plasmonic effect and quantify the improvements in terms of spectral quality associated with SERS measurements in human saliva. METHODS: Dried human saliva was characterized using spontaneous Raman spectroscopy and SERS. A fabrication protocol was implemented leading to the production of silver (Ag) nanopillar substrates by glancing angle deposition. Two different imaging systems were used to interrogate saliva from 161 healthy donors: a custom single-point macroscopic system and a Raman micro-spectroscopy instrument. Quantitative metrics were established to compare spontaneous RS and SERS measurements: the Raman spectroscopy quality factor (QF), the photonic count rate (PR), the signal-to-background ratio (SBR). RESULTS: SERS measurements acquired with an excitation energy four times smaller than with spontaneous RS resulted in improved QF, PR values an order of magnitude larger and a SBR twice as large. The SERS enhancement reached 100×, depending on which Raman bands were considered. CONCLUSIONS: Single-point measurement of dried saliva with silver nanopillars substrates led to reproducible SERS measurements, paving the way to real-time tools of diagnosis in human biofluids.

Biosynthesis of copper nanoclusters for fluorescence detection of bilirubin in biofluids.

Copper nanoclusters (Cu NCs) have shown significant attention in sensing of molecular and ionic species. In this work, a single-step biosynthetic approach was introduced for the preparation of fluorescent Cu NCs using Holarrhena pubescens (H. pubescens) leaves extract as a template. The synthesized H. pubescens-Cu NCs act as a nanomolecular probe for the detection of bilirubin in biofluids. The synthesized H. pubescens-Cu NCs displayed highest fluorescence intensity at 454 nm, when excited at 330 nm. Importantly, selective detection of bilirubin was obtained by introducing H. pubescens-Cu NCs as a simple molecular probe. The interaction of bilirubin and H. pubescens-Cu NCs resulted in a remarkable decrease in the emission peak intensity. The developed H. pubescens-Cu NCs-based bilirubin molecular probe has a wide linear range of 0.5-20.00 µM with the limit of detection of 30.54 nM for bilirubin. The promising application of H. pubescens-Cu NCs-based molecular probe was assessed by assaying bilirubin in spiked biofluids.

Fluorescence 'turn-off-on' assays for neomycin sulphate and K+ ions with orange-red fluorescent molybdenum nanoclusters.

Fluorescent metal nanoclusters (MNCs) have found extensive application in recognizing molecular species. Here, orange-red fluorescent Arg-A. paniculata-MoNCs were synthesized using Andrographis paniculata leaf extract, arginine as a ligand, and MoCl5 as a metal precursor. The Arg-A. paniculata-MoNCs complex exhibited a quantum yield (QY) of 16.91% and excitation/emission wavelengths of 400/665 nm. The synthesized Arg-A. paniculata-MoNCs successfully acted as a probe for assaying neomycin sulphate (NS) via fluorescence turn-off and K+ ions via fluorescence turn-on mechanisms, respectively. Moreover, the developed probe was effectively used to develop a cellulose paper strip-based sensor for detection of NS and K+ ions. Arg-A. paniculata-MoNCs demonstrated great potential for sensing NS and K+ ions, with concentration ranges of 0.1-80 and 0.25-110 µM for NS and K+ ions, respectively. The as-synthesized Arg-A. paniculata-MoNCs efficiently detected NS and K+ ions in food and biofluid samples, respectively.

Rapid nanomolar detection of cocaine in biofluids by electrochemical aptamer-based sensor with low-temperature effect for drugged driving screening.

Cocaine is one of the most abused illicit drugs, and its abuse damages the central nervous system and can even lead directly to death. Therefore, the development of simple, rapid and highly sensitive detection methods is crucial for the prevention and control of drug abuse, traffic accidents and crime. In this work, an electrochemical aptamer-based (EAB) sensor based on the low-temperature enhancement effect was developed for the direct determination of cocaine in bio-samples. The signal gain of the sensor at 10 °C was greatly improved compared to room temperature, owing to the improved affinity between the aptamer and the target. Additionally, the electroactive area of the gold electrode used to fabricate the EAB sensor was increased 20 times by a simple electrochemical roughening method. The porous electrode possesses more efficient electron transfer and better antifouling properties after roughening. These improvements enabled the sensor to achieve rapid detection of cocaine in complex bio-samples. The low detection limits (LOD) of cocaine in undiluted urine, 50% serum and 50% saliva were 70 nM, 30 nM and 10 nM, respectively, which are below the concentration threshold in drugged driving screening. The aptasensor was simple to construct and reusable, which offers potential for drugged driving screening in the real world.

Expression of microRNA-223 and microRNA-214 in gingival crevicular fluid of smoker and nonsmoker periodontitis patients, an observational diagnostic accuracy study.

OBJECTIVE: Periodontitis is a multifactorial disease that affects a wide range of populations. However, its pathogenesis remains unclear. miRNAs are now considered potential diagnostic markers for many inflammatory diseases. Thus, the aim of this study was to assess the expression of microRNA-223(miRNA-223) and microRNA-214 (miRNA-214) in gingival crevicular fluid (GCF) of smoker and nonsmoker with periodontitis. MATERIALS AND METHODS: We conducted a prospective study among 42 participants: 14 healthy controls, 14 nonsmoker periodontitis participants, and 14 smokers with periodontitis. Eligibility criteria for inclusion were consecutive adults, aged 20-60 years, with stage III periodontitis grade B/C and no systemic diseases. All consenting participants had gingival crevicular fluid samples collected after diagnosis to assess miRNA-214 and -223 by quantitative real-time polymerase chain reaction assay. RESULTS: ROC curve analyses for the non-smoker periodontitis group showed that miR-214 as a predictor in comparison to miR-223 had higher sensitivity [92.86%-64.29%], same specificity [100%], and a significantly higher area under the curve [0.974-0.796] respectively (p = 0.036). As for the smoker periodontitis group, a ROC curve with miR-214 as predictor in comparison to miR-223 had higher sensitivity [100%-71.43%], same specificity [100%], and a non-significantly higher area under the curve [1-0.872], respectively (p = 0.059). CONCLUSION: Both miRNA-214 and 223 are reliable potential diagnostic markers for periodontitis, with miRNA-214 being more accurate for smokers with periodontitis. CLINICAL RELEVANCE: Both miRNA-214 and 223 could be considered for potential chair-side diagnostics, by simply collecting GCF detecting the disease in its first steps and aid in preventing unrepairable damage.

Exploring the Potential of Exosomes as Biomarkers in Tuberculosis and Other Diseases.

Tuberculosis (TB) is a major cause of morbidity and mortality and remains an important public health issue in developing countries worldwide. The existing methods and techniques available for the diagnosis of TB are based on combinations of laboratory (chemical and biological), radiological, and clinical tests. These methods are sophisticated and laborious and have limitations in terms of sensitivity, specificity, and accuracy. Clinical settings need improved diagnostic biomarkers to accurately detect biological changes due to pathogen invasion and pharmacological responses. Exosomes are membrane-bound vesicles and mediators of intercellular signaling processes that play a significant role in the pathogenesis of various diseases, such as tuberculosis, and can act as promising biomarkers for the monitoring of TB infection. Compared to conventional biomarkers, exosome-derived biomarkers are advantageous because they are easier to detect in different biofluids, are more sensitive and specific, and may be useful in tracking patients' reactions to therapy. This review provides insights into the types of biomarkers, methods of exosome isolation, and roles of the cargo (proteins) present in exosomes isolated from patients through omics studies, such as proteomics. These findings will aid in developing new prognostic and diagnostic biomarkers and could lead to the identification of new therapeutic targets in the clinical setting.

Potential of in vitro nuclear magnetic resonance of biofluids and tissues in clinical research.

Body fluids, cells, and tissues contain a wide variety of metabolites that consist of a mixture of various low-molecular-weight compounds, including amino acids, peptides, lipids, nucleic acids, and organic acids, which makes comprehensive analysis more difficult. Quantitative nuclear magnetic resonance (NMR) spectroscopy is a well-established analytical technique for analyzing the metabolic profiles of body fluids, cells, and tissues. It enables fast and comprehensive detection, characterization, a high level of experimental reproducibility, minimal sample preparation, and quantification of various endogenous metabolites. In recent times, NMR-based metabolomics has been appreciably utilized in diverse branches of medicine, including microbiology, toxicology, pathophysiology, pharmacology, nutritional intervention, and disease diagnosis/prognosis. In this review, the utility of NMR-based metabolomics in clinical studies is discussed. The significance of in vitro NMR-based metabolomics as an effective tool for detecting metabolites and their variations in different diseases are discussed, together with the possibility of identifying specific biomarkers that can contribute to early detection and diagnosis of disease.

Recent advances in the detection of glioblastoma, from imaging-based methods to proteomics and biosensors: A narrative review.

Glioblastoma (GBM) is an aggressive type of cancer that originates in the cells called astrocytes, which support the functioning of nerve cells. It can develop in either the brain or the spinal cord and is also known as glioblastoma multiform. GBM is a highly aggressive cancer that can occur in either the brain or spinal cord. The detection of GBM in biofluids offers potential advantages over current methods for diagnosing and treatment monitoring of glial tumors. Biofluid-based detection of GBM focuses on identifying tumor-specific biomarkers in blood and cerebrospinal fluid. To date, different methods have been used to detect biomarkers of GBM, ranging from various imaging techniques to molecular approaches. Each method has its own strengths and weaknesses. The present review aims to scrutinize multiple diagnostic methods for GBM, with a focus on proteomics methods and biosensors. In other words, this study aims to provide an overview of the most significant research findings based on proteomics and biosensors for the diagnosis of GBM.

Standards of Fluid Biomarker Collection and Pre-analytical Processes in Humans and Mice: Recommendations by the Ataxia Global Initiative Working Group on Biomarkers.

The Ataxia Global Initiative (AGI) aims to serve as a platform to facilitate clinical trial readiness for the hereditary ataxias. Clinical trials for these diseases have been hampered by the lack of objective measures to study disease onset, progression, and treatment efficacy. While these issues are not unique to the genetic ataxias, the relative rarity of these diseases makes the need for such measures even more pressing to achieve statistical power in clinical trials. In this report, we have described the efforts of the AGI fluid biomarker working group (WG) in developing uniform protocols for biomarker sampling and storage, both for human and preclinical studies in mice. By reducing collection variability, we anticipate reduced noise in downstream biomarker analysis that will improve statistical power and minimize the necessary sample size. The emphasis has been on defining and standardizing the sampling and pre-analytical work-up of minimal set of biological samples, specifically blood plasma and serum, keeping in mind the need for harmonization of collection and storage that can be achieved with relatively limited cost and resources. An optional package is detailed for those centers that have the resources and commitment for additional biofluids/sample processing and storage. Finally, we have delineated similar standardized protocols for mice that will be important for preclinical studies in the field.

A review on comparative studies addressing exosome isolation methods from body fluids.

Exosomes emerged as valuable sources of disease biomarkers and new therapeutic tools. However, extracellular vesicles isolation with exosome-like characteristics from certain biofluids is still challenging which can limit their potential use in clinical settings. While ultracentrifugation-based procedures are the gold standard for exosome isolation from cell cultures, no unique and standardized method for exosome isolation from distinct body fluids exists. The complexity, specific composition, and physical properties of each biofluid constitute a technical barrier to obtain reproducible and pure exosome preparations, demanding a detailed characterization of both exosome isolation and characterization methods. Moreover, some isolation procedures can affect downstream proteomic or RNA profiling analysis. This review compiles and discussed a set of comparative studies addressing distinct exosome isolation methods from human biofluids, including cerebrospinal fluid, plasma, serum, saliva, and urine, also focusing on body fluid specific challenges, physical properties, and other potential variation sources. This summarized information will facilitate the choice of exosome isolation methods, based on the type of biological samples available, and hopefully encourage the use of exosomes in translational and clinical research.

Synthesis of gold and copper bimetallic nanoclusters with papain for fluorescence detection of cortisone in biological samples.

In this work, bimetallic gold and copper nanoclusters (Au-Cu NCs) were fabricated by using papain as a ligand. The as-synthesized papain-Au-Cu NCs displayed red fluorescence at 365 nm of UV lamp. The as-prepared papain-Au-Cu NCs display a strong fluorescence at 656 nm when excited at 390 nm. Papain encapsulated Au-Cu NCs exhibit distinct interaction site towards cortisone, which results red fluorescence "turn-off". Papain-Au-Cu NCs exhibited a rapid response and remarkable fluorescence quenching with the addition of cortisone as a biomarker. The developed probe is demonstrated for the quantification of cortisone by plotting the calibration graph between the fluorescence ratio (I0/I) and cortisone concentration (0.031-1.00 µM) with an impressive detection limit of 1.89 nM. Interestingly, the response of papain-Au-Cu NCs towards other biomarkers and common interfering species is quite silent, signifying the selectivity of papain-Au-Cu NCs for the detection of cortisone. The probe was successfully applied to assay cortisone biomarker in urine and plasma samples. More importantly, the analytical validation of the probe was assessed by assaying cortisone in intra- and inter-day, demonstrating papain-Au-Cu NCs could serve as promising probe for the rapid detection of cortisone in biological samples.

Updated Methods of Extracellular Vesicles Isolation.

Extracellular vesicles (EVs) are considered as cargo and mediate intercellular communication. As natural biological nanoparticles, EVs can be secreted by almost all kinds of cells and exist in biofluids such as milk, urine, blood, etc. In the past decades, several methods have been utilized to isolate EVs from cell culture medium, biofluids, and tissues. Here in this chapter, we summarized conventional and novel methods and fundamental procedures of EVs extraction and purification from different biofluids (plasma, urine, milk, and saliva) and tissues (brain, intestinal tissue, muscles, and heart). The present section also discusses how to choose appropriate methods to extract EVs from tissues based on downstream analysis. This chapter will expand the horizons of EVs isolation and purification from different mediums.

Microwave-assisted synthesis of blue fluorescent molybdenum nanoclusters with maltose-cysteine Schiff base for detection of myoglobin and γ-aminobutyric acid in biofluids.

The fabrication of stable fluorescent MoNCs (molybdenum nanoclusters) in aqueous media is quite challenging as it is not much explored yet. Herein, we report a facile and efficient strategy for fabricating MoNCs using 2,3 dialdehyde maltose-cysteine Schiff base (DAM-cysteine) as a ligand for detecting myoglobin and γ-aminobutyric acid (GABA) in biofluids with high selectivity and sensitivity. The DAM-cysteine-MoNCs displayed fluorescence of bright blue color under a UV light at 365 nm with an emission peak at 444 nm after excitation at 370 nm. The synthesized DAM-cysteine-MoNCs were homogeneously distributed with a mean size of 2.01 ± 0.98 nm as confirmed by the high-resolution transmission electron microscopy (HR-TEM). Further, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT-IR) techniques were utilized to confirm the elemental oxidation states and surface functional groups of the DAM-cysteine-MoNCs. After the addition of myoglobin and GABA, the emission peak of DAM-cysteine-MoNCs at 444 nm was significantly quenched. This resulted in the development of a quantitative assay for the detection of myoglobin (0.1-0.5 µM) and GABA (0.125-2.5 µM) with the lower limit of detection as 56.48 and 112.75 nM for myoglobin and GABA, respectively.

Biomarkers of Migraine: An Integrated Evaluation of Preclinical and Clinical Findings.

In recent years, numerous efforts have been made to identify reliable biomarkers useful in migraine diagnosis and progression or associated with the response to a specific treatment. The purpose of this review is to summarize the alleged diagnostic and therapeutic migraine biomarkers found in biofluids and to discuss their role in the pathogenesis of the disease. We included the most informative data from clinical or preclinical studies, with a particular emphasis on calcitonin gene-related peptide (CGRP), cytokines, endocannabinoids, and other biomolecules, the majority of which are related to the inflammatory aspects and mechanisms of migraine, as well as other actors that play a role in the disease. The potential issues affecting biomarker analysis are also discussed, such as how to deal with bias and confounding data. CGRP and other biological factors associated with the trigeminovascular system may offer intriguing and novel precision medicine opportunities, although the biological stability of the samples used, as well as the effects of the confounding role of age, gender, diet, and metabolic factors should be considered.