Human Biofluids Research Articles

Enhancing Nonfouling and Sensitivity of Surface-Enhanced Raman Scattering Substrates for Potent Drug Analysis in Blood Plasma via Fabrication of a Flexible Plasmonic Patch.

Surface-enhanced Raman scattering (SERS) is an ultrasensitive analytical technique, which is capable of providing high specificity; thus, it can be used for toxicological drug assay (detection and quantification). However, SERS-based drug analysis directly in human biofluids requires mitigation of fouling and nonspecificity effects that commonly appeared from unwanted adsorption of endogenous biomolecules present in biofluids (e.g., blood plasma and serum) onto the SERS substrate. Here, we report a bottom-up fabrication strategy to prepare ultrasensitive SERS substrates, first, by functionalizing chemically synthesized gold triangular nanoprisms (Au TNPs) with poly(ethylene glycol)-thiolate in the solid state to avoid protein fouling and second, by generating flexible plasmonic patches to enhance SERS sensitivity via the formation of high-intensity electromagnetic hot spots. Poly(ethylene glycol)-thiolate-functionalized Au TNPs in the form of flexible plasmonic patches show a twofold-improved signal-to-noise ratio in comparison to triethylamine (TEA)-passivated Au TNPs. Furthermore, the plasmonic patch displays a SERS enhancement factor of 4.5 ×107. Utilizing the Langmuir adsorption model, we determine the adsorption constant of drugs for two different surface ligands and observe that the drug molecules display stronger affinity for poly(ethylene glycol) ligands than TEA. Our density functional theory calculations unequivocally support the interaction between drug molecules and poly(ethylene glycol) moieties. Furthermore, the universality of the plasmonic patch for SERS-based drug detection is demonstrated for cocaine, JWH-018, and opioids (fentanyl, despropionyl fentanyl, and heroin) and binary mixture (trace amount of fentanyl in heroin) analyses. We demonstrate the applicability of flexible plasmonic patches for the selective assay of fentanyl at picogram/milliliter concentration levels from drug-of-abuse patients' blood plasma. The fentanyl concentration calculated in the patients' blood plasma from SERS analysis is in excellent agreement with the values determined using the paper spray ionization mass spectrometry technique. We believe that the flexible plasmonic patch fabrication strategy would be widely applicable to any plasmonic nanostructure for SERS-based chemical sensing for clinical toxicology and therapeutic drug monitoring.

Analysis of α-synuclein levels related to LRRK2 kinase activity: from substantia nigra to urine of patients with Parkinson’s disease

ABSTRACT Research on Parkinson’s disease (PD) has been focused on the development of PD diagnostic tools as much as the development of PD therapeutics. Several genetic culprits of PD, including DJ-1, Leucine-rich repeat kinase 2 (LRRK2), and α-synuclein (α-syn), have been investigated as markers of PD in human biofluids. Unfortunately, the approaches to develop PD diagnostic tools are impractical, and there is a considerable demand for an appropriate marker of PD. The measurement of α-syn in biofluids has recently been made more accurate by examining monomers and aggregates separately using enzyme-linked immunosorbent assay (ELISA). Previously, we reported on the development of two types of sandwich ELISA for total α-syn and MJFR-14-6-4-2 antibody-specific α-syn fibrillar oligomers. The pathogenic LRRK2 G2019S mutation is related to increased α-syn secretion in the extracellular space. We tested our established ELISA using differentiated SH-SH5Y cells transfected with LRRK2 G2019S. The secretory levels of fibrillar oligomeric α-syn divided by total α-syn were significantly increased in LRRK2 G2019S-expressing cells. Additionally, substantia nigra lysates or concentrated urine from PD patients and non-PD subjects were analyzed. We observed ambiguous changes in the levels of total or fibrillar oligomeric α-syn and their ratio between PD and non-PD. Despite the insignificant increase in the relative levels of fibrillar oligomeric α-syn to total α-syn in PD, the duration of disease progression after diagnosis significantly corresponded to the relative levels of fibrillar oligomeric α-syn to total α-syn in the urine. These results might provide greater understanding for the next stage of development of α-syn ELISAs.

Ti3c2tx-Reduced Graphene Oxide Nanocomposite Based Electrochemical Sensor for the Determination of Serotonin in Human Biofluids

Ti3c2tx-Reduced Graphene Oxide Nanocomposite Based Electrochemical Sensor for the Determination of Serotonin in Human Biofluids

D-Penicillamine functionalized dendritic fibrous nanosilica (DFNS-DPA): synthesise and its application as an innovative advanced nanomaterial towards sensitive quantification of ractopamine.

During the twentieth century, ractopamine (RAC) as one of the important and frequently used feed additives and doping agents has attracted considerable attention in the animal breeding industry and sports competitions. Due to the low metabolism rate of RAC, it is accumulated in livestock tissues. By consuming food, the residues enter the human body causing hazardous side effects including tachycardia, palpitations, and headache. So, sensitive identification of this compound is desirable to combat illicit use and protect food safety. Here, a novel nanomaterial is manufactured based on the functionalization of dendritic fibrous nanosilica with dipenicillamine (KCC-1-NH-DPA). Synthesised advanced nanomaterial was used for the encapsulation of specific DNA-aptamer and incubated on the surface of gold electrode modified by poly(β-cyclodextrin) P(β-CD) which provided the high surface area, excellent mechanical and thermal stability for the dens-loading of encapsulated aptamer. The green platform was provided an efficient apta-platform for the specific recognition of RAC in human biofluids. Electroanalysis of RAC was performed based on “signal ON” protocol. The modified gold electrode by P(β-CD)-(KCC-1-NH-DPA) was used to improve the conductivity and function of the aptasensor towards sensitive identification of RAC in human real sample. Cyclic voltammetry, differential voltammetry, square wave voltammetry, and chronoamperometry techniques were exploited for the measurement of RAC in the concentration range of 0.1 fM to 0.1 mM. Furthermore, the lower limit of quantification (LLOQ) of engineered aptasensor was obtained as 0.1 fM. It is worth noting that the proposed electrochemical aptasensor showed excellent stability, selectivity and performance in standard and human plasma samples. It is important to point out that, synergetic effect of DFNS with high surface to volume, P(β-CD) as conductive substrate and selective aptamer in the fabricated biodevice lead to highly sensitive and selective biosensor for the biomedical analysis of clinical samples. This platform will be provide a new horizon for the application of advanced nanomaterials in biomedical science based POC analysis.

Determination of sodium and potassium ions in patients with SARS-Cov-2 disease by ion-selective electrodes based on polyelectrolyte complexes as a pseudo-liquid contact phase.

Nowadays, there are several methods for the detection of various bioelements during SARS-CoV-2. Many of them require special equipment, high expenses, and a long time to obtain results. In this study, we aim to use polyelectrolyte multilayers for robust carbon fiber-based potentiometric sensing to determine the ion concentration in human biofluids of COVID-19 patients. The polyethyleneimine/polystyrene sulfonate complex is hygroscopic and has the ability to retain counterions of inorganic salts. This fact makes it possible to create a flexible ionometric system with a pseudo-liquid connection. The formation of the polyethyleneimine/polystyrene sulfonate complex allows for the adhesion of a hydrophobic ion-selective membrane, and creates a Nernst response in a miniature sensor system. This approach discloses the development of miniaturized ion-selective electrodes and their future application to monitor analyte changes as micro and macroelement ions in the human body to identify correlation to SARS-CoV-2. An imbalance in the content of potassium and sodium in urine and blood is directly related to changes in the zinc content in patients with coronavirus. The proposed method for assessing the condition of patients will allow fast determination of the severity of the course of the disease.

All-natural superhydrophobic coating for packaging and blood-repelling materials

The design of a bio-safe superhydrophobic coating to repel water adsorption, food and blood adherence is significantly important to attenuate food spoilage, food waste and thrombi. Herein, we synthesize an all-natural superhydrophobic interfacial material that has strong interfacial stability on a variety of substrates, and has good repellence towater, highly-viscous liquid foods and biofluids (blood, urine, etc.). The base material is simply prepared in an aqueous solution of lysozyme and cysteine, in which the rapid phase transition of lysozyme is triggered by a cysteine-induced thiol-disulfide exchange reaction. The resulting phase-transited lysozyme (PTL) aggregates into microparticles with a nanoscale roughness, which are further packed into a dense layer with a micro-/nano-topography. The PTL is composed of an inner amyloid-like structure and shows strong interfacial adhesion with the underlying substrate due to amyloid-inspired adhesion. The PTL layer is finally converted to a robust coating to effectively repel liquid foods and biofluids by being decorated with a natural hydrophobic carnauba wax. This coating noticeably enhances the water repellence of cellulose-based packaging materials, and the resulting superhydrophobic paper is completely non-wetted by water without weakening the mechanical stability and usability of the modified paper. The coating further resists the adhesion of milk, yogurt, honey and various other beverages and edible liquids, which leaves no residue on the coated surface. This coating also significantly resists wetting by blood and other human biofluids with a noticeable decrease in platelet adhesion on the surface. This work underlines the importance of an amyloid-like protein assembly in the preparation of an all-natural green interfacial coating towards the robust repellence of water, edible liquids and blood.

Fabrication of an electrochemical biodevice for ractopamine detection under a strategy of a double recognition of the aptamer/molecular imprinting polymer

The importance of RAC tracking in human biofluids has boosted many demands for designing an ultrasensitive tool to determine the trace value of the RAC from clinical, judicial, and forensic centers. In this study, an electrochemical biodevice has developed for the highly selective detection of this illegal feed additive under a double recognition strategy of the aptamer (Apt) and molecular imprinting polymer (MIP) on a glassy carbon electrode (GCE). The sensing relies on this fact that both the MIP and Apt act synergistically to trap the RAC molecules. The sensing surface fabrication steps have been monitored by some electrochemical techniques such as electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV(. The charge transfer resistance (Rct) value of the redox probe as a representative of the biodevice response has increased linearly with the RAC concentration increasing in a dynamic range of 1 fM to 1.90 µM. The detection limit (LOD) value has been estimated to be 330 aM, lower than all of the reported methods in the RAC sensing. Furthermore, the practical feasibility of biodevice has been evaluated in some human blood serum and urine samples. This strategy offers some useful advantages in reliable detection of the RAC, which may help in the routine analysis, as mandated by regulatory agencies.

Non-Coding RNAs as Blood-Based Biomarkers in Cardiovascular Disease.

In 2020, cardiovascular diseases (CVDs) remain a leading cause of mortality and morbidity, contributing to the burden of the already overloaded health system. Late or incorrect diagnosis of patients with CVDs compromises treatment efficiency and patient’s outcome. Diagnosis of CVDs could be facilitated by detection of blood-based biomarkers that reliably reflect the current condition of the heart. In the last decade, non-coding RNAs (ncRNAs) present on human biofluids including serum, plasma, and blood have been reported as potential biomarkers for CVDs. This paper reviews recent studies that focus on the use of ncRNAs as biomarkers of CVDs.

Sensitive, Rapid, Low-Cost, and Multiplexed COVID-19 Monitoring by the Wireless Telemedicine Platform

To prevent the more severe spread of COVID-19 infections, sensitive, rapid, low-cost, and multiplexed detection is critical. Recently, Gao et al. reported a laser-engraved graphene-based wireless device to monitor multiple biomarkers from human biofluids, allowing for high-frequency self-testing of COVID-19 with high accuracy and low cost.

Charting Extracellular Transcriptomes in The Human Biofluid RNA Atlas

Extracellular RNAs present in biofluids have emerged as potential biomarkers for disease. Where most studies focus on blood-derived fluids, other biofluids may be more informative. We present an atlas of messenger, circular, and small RNA transcriptomes of a comprehensive collection of 20 human biofluids. By means of synthetic spike-in controls, we compare RNA content across biofluids, revealing a 10,000-fold difference in concentration. The circular RNA fraction is increased in most biofluids compared to tissues. Each biofluid transcriptome is enriched for RNA molecules derived from specific tissues and cell types. Our atlas enables an informed selection of the most relevant biofluid to monitor particular diseases. To verify the biomarker potential in these biofluids, four validation cohorts representing a broad spectrum of diseases were profiled, revealing numerous differential RNAs between case and control subjects. Spike-normalized data are publicly available in the R2 web portal for further exploration.

Mass spectrometric studies on the peptide integrity of substance P and related human tachykinins in human biofluids

The neurokinin-1 receptor plays a profound role in inflammatory processes and is involved in immune cell differentiation, cytokine release, and mast cell activation. Due to their similar peptide structures, the neurokinin-1 receptor does not discriminate between the endogenous ligands substance P (SP) and human hemokinin-1 (hHK-1), which both demonstrate biological receptor affinity. In addition, due to cross-reactivity, the current bioanalytical method of choice–immunoassays–also displays limitations in differentiating between these peptides. Thus, a recently developed mass spectrometric assay was utilized for the selective quantification of SP and hHK-1 in various biofluids and tissue. By applying the sample processing protocols developed, SP was quantified in porcine brain tissue (4.49 ± 0.53 nM), human saliva (113.3 ± 67.0 pM), and human seminal fluid (0.52 ± 0.15 nM) by mass spectrometric analysis. As previously reported, neither SP nor hHK-1 could be detected in human plasma by mass spectrometry. Comparison with analysis using a commercial immunoassay of the same plasma sample revealed SP like-immunoreactivity concentrations of 37.1–178.0 pM. The previously reported carboxylic acid of SP, whose identity was confirmed by high-resolution mass spectrometric analysis, did not show cross-reactivity in the applied immunoassay and did not contribute to SP-like immunoreactivity results. Subsequent compound discovery of the immunocaptured substance indicated the presence of a precursor of SP as possible cross-reactor in human plasma samples. The found cross-reactivity might be the cause for the high variance of SP plasma levels in former determinations.

Circulating 16S RNA in Biofluids: Extracellular Vesicles as Mirrors of Human Microbiome?

The human body is inhabited by around 1013 microbes composing a multicomplex system, termed microbiota, which is strongly involved in the regulation and maintenance of homeostasis. Perturbations in microbiota composition can lead to dysbiosis, which has been associated with several human pathologies. The gold-standard method to explore microbial composition is next-generation sequencing, which involves the analysis of 16S rRNA, an indicator of the presence of specific microorganisms and the principal tool used in bacterial taxonomic classification. Indeed, the development of 16S RNA sequencing allows us to explore microbial composition in several environments and human body districts and fluids, since it has been detected in “germ-free” environments such as blood, plasma, and urine of diseased and healthy subjects. Recently, prokaryotes showed to generate extracellular vesicles, which are known to be responsible for shuttling different intracellular components such as proteins and nucleic acids (including 16S molecules) by protecting their cargo from degradation. These vesicles can be found in several human biofluids and can be exploited as tools for bacterial detection and identification. In this review, we examine the complex link between circulating 16S RNA molecules and bacteria-derived vesicles.

Tandem reassembly of split luciferase-DNA chimeras for bioluminescent detection of attomolar circulating microRNAs using a smartphone

Detection of dysregulated circulating microRNAs (miRNAs) in human biofluids is a fundamental ability to determine tumor occurrence and metastasis in a minimally invasive fashion. However, the requirements for sophisticated instruments and professional personnel impede the translation of miRNA tests into routine clinical diagnostics, especially for resource-limited regions. Herein, we developed a DNA-guided bioluminescence strategy for the detection of circulating miRNAs. In this strategy, a pair of split luciferase-DNA chimeras was constructed and integrated into the miRNA-triggered rolling circle amplification (RCA) process. The tandem reassembly of split luciferase-DNA chimeras on the RCA products elicited a turn-on bioluminescence response with ultrahigh signal-to-background (S/B) ratio. This strategy enabled smartphone-based assays for different miRNAs with attomolar sensitivity and single-base specificity, as demonstrated here for miR-21. miR-148b, and cel-miR-39. Further application of our approach to the clinical serum samples realized identification of dysregulated miR-21 and miR-148b in the lung cancer patients, showing a satisfactory agreement with the control assays performed with quantitative reverse transcription polymerase chain reaction (qRT-PCR). Therefore, the developed method possesses the benefits of high performance and reliability, offering a potential tool for implementing miRNA-based diagnosis in point-of-care (POC) settings.

Prediction of liquid chromatographic retention time using quantitative structure-retention relationships to assist non-targeted identification of unknown metabolites of phthalates in human urine with high-resolution mass spectrometry

The non-targeted analysis and identification of contaminant metabolites such as metabolites of phthalates and their alternatives in human biofluid samples constitutes a growing research field in human biomonitoring because of their importance as biomarkers of human exposure to the parent compounds. High-resolution mass spectrometry (HRMS) combined with high-performance liquid chromatography (HPLC) can provide fast separation and sensitive analysis using this application. However, the diversity of potential metabolites, especially isomers, in human samples, makes mass spectrometry-based structural identification very challenging, even with high-resolution and accurate mass. In this study, we present a retention time (tR) prediction model based on quantitative structure-retention relationship (QSRR). This model can predict the retention time of a given structure of phthalates including isomers. Twenty-three molecular descriptors were used in the development of the multivariate linear regression QSRR model. The regression coefficient (R2) between predicted and experimental retention times of 26 training set compounds was 0.9912. The combination of the retention time prediction model with identification via accurate mass search and target MS/MS spectrum interpretation can enhance the identification confidence in the lack of reference standards. Two previously unreported phthalate metabolites were identified in human urine, using this model. The results of this study showed that the developed QSRR model could be a useful tool to predict the retention times of unknown metabolites of phthalates and their alternatives in future non-targeted screening analysis. The concentration of these two unknown compounds was also estimated using a quantitative structure–ion intensity relationship (QSIIR) model.

A simultaneous exploratory and quantitative amino acid and biogenic amine metabolic profiling platform for rapid disease phenotyping via UPLC-QToF-MS

Metabolic phenotyping using mass spectrometry (MS) is being applied to ever increasing sample numbers in clinical and epidemiology studies. High-throughput and robust methods are being developed for the accurate measurement of metabolites associated with disease. Traditionally, quantitative assays have utilized triple quadrupole (QQQ) MS based methods; however, the use of such focused methods removes the ability to perform discovery-based metabolic phenotyping. An integrated workflow for the hybrid simultaneous quantification of 34 biogenic amines in combination with full scan high-resolution accurate mass (HRAM) exploratory metabolic phenotyping is presented. Primary and secondary amines are derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate prior to revered-phase liquid chromatographic separation and mass spectrometric detection. Using the HRAM-MS data, retrospective phenotypic data mining could be performed, demonstrating the versatility of HRAM-MS instrumentation in a clinical and molecular epidemiological environment. Quantitative performance was assessed using two MS detector platforms: Waters TQ-XS (QQQ; n = 3) and Bruker Impact II QToF (HRAMS-MS; n = 2) and three human biofluids (plasma, serum and urine). Finally, each platform was assessed using a certified external reference sample (NIST SRM 1950 plasma). Intra- and inter-day accuracy and precision were comparable between the QQQ and QToF instruments (<15%), with excellent linearity (R2 > 0.99) over the quantification range of 1–400 μmol L−1. Quantitative values were comparable across all instruments for human plasma, serum and urine samples, and calculated concentrations were verified against certified reference values for NIST SRM 1950 plasma as an external reference. As a real-life biological exemplar, the method was applied to plasma samples obtained from SARS-CoV-2 positive patients versus healthy controls. Both the QQQ and QToF approaches were equivalent in being able to correctly classify SARS-CoV-2 positivity. Critically, the use of HRAM full scan data was also assessed for retrospective exploratory mining of data to extract additional biogenic amines of biomarker interest beyond the 34 quantified targets.

Simultaneous Isolation of Circulating Nucleic Acids and EV-Associated Protein Biomarkers From Unprocessed Plasma Using an AC Electrokinetics-Based Platform.

The power of personalized medicine is based on a deep understanding of cellular and molecular processes underlying disease pathogenesis. Accurately characterizing and analyzing connections between these processes is dependent on our ability to access multiple classes of biomarkers (DNA, RNA, and proteins)—ideally, in a minimally processed state. Here, we characterize a biomarker isolation platform that enables simultaneous isolation and on-chip detection of cell-free DNA (cfDNA), extracellular vesicle RNA (EV-RNA), and EV-associated proteins in unprocessed biological fluids using AC Electrokinetics (ACE). Human biofluid samples were flowed over the ACE microelectrode array (ACE chip) on the Verita platform while an electrical signal was applied, inducing a field that reversibly captured biomarkers onto the microelectrode array. Isolated cfDNA, EV-RNA, and EV-associated proteins were visualized directly on the chip using DNA and RNA specific dyes or antigen-specific, directly conjugated antibodies (CD63, TSG101, PD-L1, GPC-1), respectively. Isolated material was also eluted off the chip and analyzed downstream by multiple methods, including PCR, RT-PCR, next-generation sequencing (NGS), capillary electrophoresis, and nanoparticle size characterization. The detection workflow confirmed the capture of cfDNA, EV-RNA, and EV-associated proteins from human biofluids on the ACE chip. Tumor specific variants and the mRNAs of housekeeping gene PGK1 were detected in cfDNA and RNA isolated directly from chips in PCR, NGS, and RT-PCR assays, demonstrating that high-quality material can be isolated from donor samples using the isolation workflow. Detection of the luminal membrane protein TSG101 with antibodies depended on membrane permeabilization, consistent with the presence of vesicles on the chip. Protein, morphological, and size characterization revealed that these vesicles had the characteristics of EVs. The results demonstrated that unprocessed cfDNA, EV-RNA, and EV-associated proteins can be isolated and simultaneously fluorescently analyzed on the ACE chip. The compatibility with established downstream technologies may also allow the use of the platform as a sample preparation method for workflows that could benefit from access to unprocessed exosomal, genomic, and proteomic biomarkers.

Extracellular microRNA 3’ end modification across diverse body fluids

ABSTRACT microRNAs (miRNAs) are small non-coding RNAs that play critical roles in gene regulation. The presence of miRNAs in extracellular biofluids is increasingly recognized. However, most previous characterization of extracellular miRNAs focused on their overall expression levels. Alternative sequence isoforms and modifications of miRNAs were rarely considered in the extracellular space. Here, we developed a highly accurate bioinformatic method, called miNTA, to identify 3’ non-templated additions (NTAs) of miRNAs using small RNA-sequencing data. Using miNTA, we conducted an in-depth analysis of miRNA 3’ NTA profiles in 1047 extracellular RNA-sequencing data sets of 4 types of biofluids. This analysis identified hundreds of miRNAs with 3’ uridylation or adenylation, with the former being more prevalent. Among these miRNAs, up to 53% (22%) had an average 3’ uridylation (adenylation) level of at least 10% in a specific biofluid. Strikingly, we found that 3’ uridylation levels enabled segregation of different types of biofluids, more effectively than overall miRNA expression levels. This observation suggests that 3’ NTA levels possess fluid-specific information relatively robust to batch effects. In addition, we observed that extracellular miRNAs with 3’ uridylations are enriched in processes related to angiogenesis, apoptosis, and inflammatory response, and this type of modification may stabilize base-pairing between miRNAs and their target genes. Together, our study provides a comprehensive landscape of miRNA NTAs in human biofluids, which paves way for further biomarker discoveries. The insights generated in our work built a foundation for future functional, mechanistic, and translational discoveries.

Aging markers in human urine: A comprehensive, non-targeted LC-MS study.

Metabolites in human biofluids document the physiological status of individuals. We conducted comprehensive, non‐targeted, non‐invasive metabolomic analysis of urine from 27 healthy human subjects, comprising 13 young adults (30 ± 3 years) and 14 seniors (76 ± 4 years). Quantitative analysis of 99 metabolites revealed 55 that displayed significant differences in abundance between the two groups. Forty‐four did not show a statistically significant relationship with age. These include 13 standard amino acids, 5 methylated, 4 acetylated, and 9 other amino acids, 6 nucleosides, nucleobases, and derivatives, 4 sugar derivatives, 5 sugar phosphates, 4 carnitines, 2 hydroxybutyrates, 1 choline, and 1 ethanolamine derivative, and glutathione disulfide. Abundances of 53 compounds decreased, while 2 (glutathione disulfide, myo‐inositol) increased in elderly people. The great majority of age‐linked markers were highly correlated with creatinine. In contrast, 44 other urinary metabolites, including urate, carnitine, hippurate, and betaine, were not age‐linked, neither declining nor increasing in elderly subjects. As metabolite profiles of urine and blood are quite different, age‐related information in urine offers additional valuable insights into aging mechanisms of endocrine system. Correlation analysis of urinary metabolites revealed distinctly inter‐related groups of compounds.

Sensitive monitoring of doxorubicin in plasma of patients, MDA-MB-231 and 4T1 cell lysates using electroanalysis method

In the present work, an innovative electrochemical sensor was fabricated based on poly toluidine blue modified glassy carbon electrode (PTB-GCE). So, PTB-GCE was used for the detection and determination of doxorubicin hydrochloride (DOX) in cell lysate, and whole human plasma samples. PTB could enhance the rate of electrochemical reaction for the electro oxidation and detection of DOX in real samples. Cyclic voltammetry (CV) technique was used for the electro polymerization of toluidine blue on the surface of GCE with the applied potential ranging from -0.6 to 0.2 V. The sensor construction steps were approved by field emission scanning electron microscopy (FE-SEM), Energy dispersive X-ray spectroscopy (EDX) and electrochemical methods. Also, CV results indicated that the DOX is oxidized via two electrons and two protons process at the optimum pH of 6.5 using PTB modified GCE. Under optimized conditions, differential pulse voltammetry (DPV) technique response exhibited linear relationship between the oxidative peak current and concentration of DOX in the range of 17 nM - 8.6 μM with low limit of quantification (LLOQ) of 17 nM for untreated and treated human plasma samples. Also, determination of DOX in MDA-MB-231 and 4T1cell lysates were performed based on its direct electrochemical oxidation on PTB-GCE. Finally, analytical validation of DOX in human bio-fluids using FDA guideline were done successfully. Results suggested that the proposed electrochemical sensor can be used to the sensitive and selective determination of DOX in biological samples. The interaction results of DOX with cancer cells indicate the developed probe can easily detect candidate drug in cancer cells with high accuracy. To the best of our knowledge this is the first report of the determination of DOX based on the direct electrochemical oxidation on PTB-GCE and determination in MDA-MB-231 and 4T1 cell lysates. It is anticipated that this research open new horizons on the design of new class of electrochemical sensors for determination drugs, and therapeutic drug monitoring (TDM) in human bio-fluids.

A Flexible and Regenerative Aptameric Graphene–Nafion Biosensor for Cytokine Storm Biomarker Monitoring in Undiluted Biofluids toward Wearable Applications

AbstractWearable sensors that can conveniently detect cytokine levels in human biofluids are essential for assisting hospitals to maximize the benefits of anti‐inflammatory therapies and avoid cytokine storms. Measurement of cytokine levels in biofluids still remains challenging for existing sensors due to high interference from the background. Here, this challenge is overcome through developing a flexible and regenerative aptameric field‐effect transistor biosensor, consisting of a graphene–Nafion composite film, for detecting cytokine storm biomarkers in undiluted human biofluids. The composite film enables the minimization of nonspecific adsorption and empowers the renewability to the biosensor. With these capabilities, the device is capable of consistently and sensitively monitoring cytokines (e.g., IFN‐γ, an inflammatory and cancer biomarker) in undiluted human sweat with a detection range from 0.015 to 250 nm and limit of detection down to 740 fm. The biosensor is also shown to incur no visible mechanical damage and maintain a consistent sensing response throughout the regenerative (up to 80 cycles) and crumpling (up to 100 cycles) tests. Experimental results demonstrate that the biosensor is expected to offer opportunities for developing wearable biosensing systems for distinguishing acute infectious disease patients and monitoring of patients’ health conditions in daily life.