Human Saliva Samples Research Articles

1881. Detection of SARS-CoV-2 Transmission on a College Campus using a Combined Contact Tracing and Molecular Strategy

Abstract Background SARS-CoV-2 has caused unprecedented global disruption following its introduction into the human population. Beginning in August 2021, a residential college initiated an asymptomatic testing protocol using PCR-based detection of human saliva samples (Thermofisher, TaqPath SARS-CoV-2 Assay; QuantStudio v1.3). In January 2021 all students were invited to campus and were required to test once or twice weekly in an effort to isolate those positive (cases) as early as possible and minimize transmission. Methods Cases were contacted within 12 hours by trained clinical staff and interviewed with a standardized questionnaire to determine contacts and exposures. Positive samples were submitted for RNA-Seq analysis (ARTIC amplicon sequencing protocol, Illumina MiSeq) and analyzed using Nextclade and USHER (comparison data from GISAID). Using this sequence data, we monitored the evolution, transmission, and emergence of variants over time in the campus community. Results In March 2022 a one-week break and the activities preceding it were followed by an outbreak of COVID among campus members. All cases were attributed to the Omicron variant. Based on traditional contact tracing 10 presumed clusters of transmission were identified (34 students). Sequence data from these 34 samples were assembled to identify phylogenetic and molecular patterns of similarity. Several molecular signatures were identified. In a group of 5 cases, all were Omicron BA.2.9; these individuals comprised a clinical cluster of students who had travelled together. Other lineages were BA.2 (N=26), BA.1(N=2), BA.2.12. (N=3), BA.2.3 (N=1) and an Omicron Clade 21M variant that appeared to be a BA.1/BA.2 recombinant (N=2). The BA.2.12 samples were contributed over 2 days; they were found in two different clusters (a social club and an artistic group), demonstrating likely transmission between members. The BA.2 samples were distributed across a wide number of student groups, and while not identical with one another, did not fall into the clusters identified by contact tracing, suggesting endemic transmission across our campus. Conclusion This analysis demonstrates that molecular analysis of SARS-CoV-2 transmission can supplement and inform the data provided by clinical/epidemiological analysis of cases. Disclosures All Authors: No reported disclosures.

Impact of DNA Extraction Methods on Quantitative PCR Telomere Length Assay Precision in Human Saliva Samples

Telomere length (TL) has emerged as a promising replicative cellular aging marker that reflects both genetic and non-genetic influences. Quantitative PCR (qPCR) TL measurement has been favored as a cost-effective method that can be easily implemented, especially in population studies with limited quantities of source material. However, several recent reports have revealed inconsistencies in telomere length measurements when applying different DNA extraction methods to the same source material. In this study we tested three DNA extraction methods on saliva samples from 48 participants of the National Growth and Health Study (NGHS) collected with DNA Genotek’s Oragene kit. The chosen extraction kits represent three distinct approaches to genomic DNA extraction from lysed cells and we employed two different operators to carry out all assays on the same samples. We measured DNA yield and quality and calculated the between-operator agreement of qPCR TL measurements (intraclass correlation, ICC). Our analyses showed that while both QIAamp and Agencourt DNAdvance had higher agreement between the 2 operators (ICC=0.937, CI [0.891, 0.965] and ICC=0.95, CI [0.911, 0.972] respectively), compared to PrepIT kit (ICC=0.809, CI [0.678, 0.889]), QIAamp extracted DNA samples were notably degraded. Using generalizability theory, we found that the participant-by-extraction-method interaction explained about 10% of total variation in TL, suggesting that TL differences across methods are somewhat participant-specific. Therefore, our results suggest that the among the three DNA extraction methods tested, Agencourt (magnetic bead purification) is the preferred kit, and we also strongly recommend against combining different extraction methods within a study population.

Disposable screen-printed electrochemical sensing strips for rapid decentralized measurements of salivary ketone bodies: Towards therapeutic and wellness applications

The interest in ketone bodies (KBs) has intensified recently as they play significant roles in healthcare, nutrition, and wellness applications. We present a disposable electrochemical sensing strip for rapid decentralized detection of β-hydroxybutyrate (HB), one of the dominant physiological KBs, in saliva. The new salivary enzymatic HB sensor strip relies on a gold-coated screen-printed carbon electrode modified with a reagent layer containing toluidine blue O (TBO mediator), β-hydroxybutyrate dehydrogenase (HBD enzyme), and the HBD cofactor nicotinamide adenine dinucleotide (NAD+ coenzyme), along with carbon nanotubes (CNTs) and chitosan (Chit) for enhancing the sensor's sensitivity and for encapsulating the enzyme and its cofactor, respectively. The systematic optimization resulted in an attractive analytical performance, with a rapid response time within 60 s, a wide HB dynamic detection range from 0.1 to 3.0 mM along with a low limit of detection (50 μM HB) in an artificial saliva medium. The strip displays high selectivity for HB over acetoacetate (AcAc) and other interferences (i.e., acetaminophen, ascorbic acid, glucose, lactic acid, and uric acid), good reproducibility, and high stability towards temperature or pH effects. The new disposable sensing strip system, coupled with a hand-held electrochemical analyzer, showed rapid HB monitoring in human saliva samples collected from healthy volunteers, with similar temporal profiles to those obtained in parallel capillary blood measurements in response to the intake of keto supplements. This strip enables efficient, reliable, and near real-time salivary HB detection to track non-invasively the dynamics of HB concentrations after intaking commercial supplements towards diverse healthcare and nutrition applications.

Imprinted ratiometric fluorescence capillary sensor based on UiO-66-NH2 for rapid determination of sialic acid

Development of method with micro-volume reagent usage and high selectivity for rapid determination of valuable disease-related biomarkers is of great importance for disease surveillance. Herein, a novel ratiometric fluorescence capillary imprinted sensor was developed for highly selective fluorescence determination of sialic acid (SA) based on luminescent metal-organic framework (UiO-66-NH2). In this strategy, the imprinted pre-polymer with double emissions was sucked into the capillary to grow imprinted layer on the inner wall of the capillary. In presence of SA, the blue fluorescence (440 nm) served as fluorescence response signal was quenched due to the electron transfer mechanism between metal organic framework and SA while the red fluorescence (655 nm) served as the reference signal was remained. Within SA concentration linear range of 1.0 × 10−7–3.0 × 10−4 M, the ratiometric fluorescence capillary imprinted sensor was applied to detect SA in human serum and saliva samples successfully with a detection limit of 2.5 × 10−8 M. A sensitive and selective fluorescence method with micro-volume sample consumption (15 μl) for determination of SA in practical samples was established.

Development of magnetic molecularly imprinted polymers for the extraction of salivary pepsin prior to analysis by a novel HPLC-SEC method

Salivary pepsin is a very important and selective biomarker for gastroesophageal reflux disease and high concentrations in the saliva of potential patients can provide a very useful and non-invasive diagnostic tool. Therefore, this study focused on the development of new and highly selective magnetic molecularly imprinted polymers (MIPs) for the extraction of pepsin from human saliva samples. The developed MIPs provide a stable, cost-effective, and reusable synthetic method of extraction for pepsin. Surface imprinting technique was applied for the synthesis of MIPs to provide a wider surface area and faster binding/release of pepsin, since all the binding sites are allocated on the surface of iron oxide nanoparticles. Methacrylic acid was used as functional monomer, ethylene glycol dimethacrylate as cross linker, and deionised water as a porogen. The imprinting procedure was meticulously optimised to reach the highest possible binding indicated by the value of binding capacity Q which reached (190 mg g−1) and an imprinting factor of 1.34, calculated in comparison to non-imprinted polymers (NIPs). Moreover, the surface morphology, thermal stability as well as the binding properties were characterised to ensure validity. The developed MIPs were applied successfully for dispersive solid phase extraction of pepsin from saliva samples. Consequently, a novel, highly sensitive and simple HPLC-SEC method was developed for the quantitation of pepsin fragments extracted using MIPs. The method was optimised and validated according to ICH guidelines where the linearity ranged from 5 to 150 μg mL−1 in saliva samples with LOD reaching down to 0.6 μg mL−1 which is comparable to the low concentrations of pepsin expected to be found in real saliva samples. The developed system enables the accurate extraction and determination of salivary pepsin with recoveries reaching to 98%. Therefore, this system shows potential as an efficient and reliable testing method for pepsin.

A new ultrasensitive platform based on f-GCNFs@nano-CeO2 core-shell nanocomposite for electrochemical sensing of oxidative stress biomarker 3-nitrotyrosine in presence of uric acid and tyrosine

Electrode materials with high sensitivity and selectivity play a central role in the fabrication of electrochemical sensing platforms. In this work, functionalized graphitized carbon nanofibers (f-GCNFs)core and nano-sized CeO2 (nano-CeO2)shell was synthesized. The XRD, SEM, HRTEM and the EDX results confirmed the formation of the f-GCNFs@nano-CeO2 core–shell nanocomposite. To construct the sensor, the graphite rod electrode (GRE) has been modified with f-GCNFs@nano-CeO2. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) results revealed that the charge transfer on the f-GCNFs@nano-CeO2/GRE was dramatically enhanced compared to the other electrodes. The prepared platform (f-GCNFs@nano-CeO2) exhibited high electro-catalytic activity and the strong synergistic activity between f-GCNFs and nano-CeO2 increased the electroactive surface area. The fabricated f-GCNFs@nano-CeO2/GRE sensor has been utilized for the detection of the oxidative stress biomarker nitrotyrosine (NTyr) using SW-AdASV. The LOD and the LOQ are found to be 0.86 and 2.9 nM, respectively, with the dynamic range 2.0–1720 nM and sensitivity of 145 μAμM−1 cm−2. The developed sensor is highly selective for NTyr detection in the presence of uric acid (UA) and tyrosine (Tyr). Moreover, the fabricated sensor also shows good selectivity, favorable reproducibility, and long-term stability. The applicability of the fabricated sensor demonstrated excellent recovery of NTyr in spiked human serum, urine, and saliva samples. In the end, f-GCNFs@nano-CeO2/GRE was applied to the determination of UA, NTyr, and Tyr simultaneously.

Electrochemical Sensing of Cortisol in Human Saliva and Serum Using DNA-Steroid Conjugation with a Versatile DNA Nanostructure Sensor

Having a generalizable point of care (POC) method for clinically relevant biomolecules would greatly enhance healthcare management and disease diagnosis. Recently our group developed a novel DNA nanostructure architecture for versatile detection of analytes which is generalizable for assaying several small molecules and their larger protein binding partners (e.g. antibodies) in human serum. The DNA nanostructure is built through on-electrode enzymatic ligation of three oligos for electrode attachment, anchor binding, and electrochemical signaling. In this study, we developed an economical synthetic approach for making oligonucleotide-steroid conjugates, and we explored the capability of these DNA nanostructure sensors for small molecule/steroid detection.Cortisol is a steroid hormone secreted by the hypothalamic-pituitary-adrenal system in response to the body’s stress level. As the main stress hormone, it controls processes such as immune, adrenal, circulatory, and metabolic. Moreover, anomalies of cortisol levels can result in serious conditions such as Addison’s disease, Cushing’s syndrome, and adrenal insufficiencies. Every year, 120,000 deaths are attributed, in part, to elevated levels of stress. To minimize these issues, a simplified method for cortisol sensing is vital. In this study, we conjugated cortisol to amine-tagged DNA and used the conjugates in our sensors. Anti-cortisol antibodies induced a 67% signal drop, validating conjugation. A calibration curve for cortisol showed a limit of detection of 800 pM and a dynamic range of 1 – 100 nM. The sensor was validated in saliva and human serum samples using the gold standard method, ELISA. Changing cortisol levels in two human patients' saliva samples were successfully detected after several collection times throughout a single day. As a potential point-of-care (POC) detection device, our novel cortisol biosensor could detect the hormone in human serum and saliva samples within 6 minutes.

Low-Cost Printed Point-of-Care Electrochemical Sensors for Detecting Cortisol

Screen printing system has been used as one of the important technologies for developing ready to use portable biosensor devices. Screen printing allows the batch production of sensors on flexible substrates, textile materials and other wearable accessories. Owing to an exceptional performance such as flexibility, low cost, simple fabrication, and ability to translated into industrial scale production, screen printing technique advances the marketing potential of the electrochemical sensors. Unlike the advanced printing techniques, the screen-printing technique is less dependent on surface charge, particle size and the viscosity of the printing ink. In this work, we have attempted to develop a flexible electrochemical sensor for detecting cortisol levels using screen printing technique. Batches of three electrode sensor systems were designed and printed in uniform dimensions (2 cm x 2 cm). The mask for sensor design is fabricated using a transparent polyester sheet with an aid of XY cutter/plotter. Graphene ink was used to create working and counter electrodes, while the Ag/AgCl ink was used to create the reference electrode. A homemade silver ink stabilized with hydrogel network was used to create conductive tracks. A reagent-free detection of cortisol was demonstrated using a redox probe (methylene blue) tagged cortisol specific aptamer immobilized onto the working electrode. The variability between the sensor batches is also investigated. The functionality of the printed sensor was demonstrated for monitoring cortisol levels in human saliva samples. The printed sensing platform can also be integrated with wearable textile platform for monitoring cortisol levels in sweat.

30-Seconds Sars-Cov-2 Human Sample Diagnosis and Analytical Specificity Analysis Using Disposable Strips on a Metal-Oxide-Semiconductor Field-Effect Transistor Platform

A SARS-CoV-2 rapid detection technology employed commercially available test strips similar to the blood glucose test strips were developed with a detection time of 30 sec and the sensitivity as good as polymerase chain reaction (PCR). The analytical specificity of this technology was also investigated by testing the cross-reactivity and microbial interference on this biosensor platform using FDA suggested 31 different contaminants. Variants of SARS-CoV-2 have mutated along with the worldwide spread of the pandemic. Currently, the most common detection methods are PCR and lateral flow tests. The former takes more than an hour to obtain results and the latter has difficulty detecting the virus at low concentrations (for cycling threshold values, CT values, >25 to 27). Therefore, the demand for a fast, cost-effective, and low detection limit testing method has been significantly increased. In this work, 60 human saliva samples with 30 positive and 30 negative were collected and tested with PCR for SARS-CoV-2 prior to using our disposable strips. For this technology, the test strip was connected to the gate electrode of the MOSFET on the printed circuit board that serves to amplify signals. A synchronous double-pulsed bias voltages (around 1 ms) were sent to both the drain and gate of the MOSFET. The resulting change in drain waveforms was converted to digital readings and compared with cycling threshold (Ct) values of human samples to assess this sensor technology. The result signifies that positive human samples with a range of Ct values from 17.8 to 40 can be differentiated, along with proving none of those high-risk organisms would hinder the sensitivity of our system. Which demonstrated the potential of this system to be developed into a cost-effective and point-of-care rapid detection for SARS-CoV-2. Figure 1

A Core-Shell Au@TiO2 and Multi-Walled Carbon Nanotube-Based Sensor for the Electroanalytical Determination of H2O2 in Human Blood Serum and Saliva.

A hydrogen peroxide (H2O2) sensor was developed based on core–shell gold@titanium dioxide nanoparticles and multi-walled carbon nanotubes modified glassy carbon electrode (Au@TiO2/MWCNTs/GCE). Core–shell Au@TiO2 material was prepared and characterized using a scanning electron microscopy and energy dispersive X-ray analysis (SEM/EDX), transmission electron microscopy (TEM), atomic force microscopy (AFM), Raman spectroscopy, X-ray diffraction (XRD) and Zeta-potential analyzer. The proposed sensor (Au@TiO2/MWCNTs/GCE) was investigated electrochemically using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The analytical performance of the sensor was evaluated towards H2O2 using differential pulse voltammetry (DPV). The proposed sensor exhibited excellent stability and sensitivity with a linear concentration range from 5 to 200 µM (R2 = 0.9973) and 200 to 6000 µM (R2 = 0.9994), and a limit of detection (LOD) of 1.4 µM achieved under physiological pH conditions. The practicality of the proposed sensor was further tested by measuring H2O2 in human serum and saliva samples. The observed response and recovery results demonstrate its potential for real-world H2O2 monitoring. Additionally, the proposed sensor and detection strategy can offer potential prospects in electrochemical sensors development, indicative oxidative stress monitoring, clinical diagnostics, general cancer biomarker measurements, paper bleaching, etc.

Pullulan-stabilized gold nanoparticles tablet as a nanozyme sensor for point-of-care applications

In this article, we first report the fabrication of pullulan-stabilized gold nanoparticles tablet (AuNPs-pTab) as a point-of-care (POC) analytical device using pullulan-AuNPs solution (AuNPs-pSol) without any additional tablet forming ingredient and then use AuNPs-pTab as a colorimetric sensor for glucose detection in human saliva samples. The AuNPs-pTab sensor fabrication comprises three steps: 1) synthesis of AuNPs-pSol, 2) formation of AuNPs-pTab in an oven at 50 °C for 30 min, and 3) colorimetric detection of glucose using AuNPs-pTab as peroxidase-mimic enzymatic activity employing TMB-H2O2 system. AuNPs-pSol was synthesized by one-pot two-steps method using gold (III) chloride and pullulan as an all-in-one reagent; reducing/stabilizing/tablet forming agent followed by casting of AuNPs-pTab. The fabricated tablet was characterized using UV–vis, FTIR, TEM, DLS, and zeta potential analysis, and results were compared with AuNPs-pSol. The morphology of nanoparticles in tablet was similar to the AuNPs in solution phase. The AuNPs-pTab is stable for >6 months as compared to AuNPs-pSol which lost stability after one month. The peroxidase-mimic enzymatic behavior of AuNPs-pTab was 25% better than AuNPs-pSol as depicted by the development of intense blue color of oxidized TMB. The effect of pH, temperature, and concentration of TMB as well as AuNPs-pTab was studied to see their impact on glucose assay. The reaction rate was calculated using Michaelis Menten's model of TMB oxidation while the calibration curve for glucose monitoring is fitted using a dose-response equation with a linear range of 0–5 μM of glucose. The limit of detection (LoD) for H2O2 was found to be 28.7 μM in the buffer while it was 38.2 and 163.04 μM for glucose in buffer and artificial human saliva, respectively. Percentage recovery for real human saliva was calculated as 93.6–107.7%. Our newly proposed AuNPs-pTab sensor has shown excellent peroxidase-like activity and provides an easy substitute for AuNPs solution with enhanced catalytic efficiency. Moreover, the AuNPs-pTab sensor is a promising platform for POC devices due to its fulfillment of ASSURED criteria. AuNPs-pTab sensor opens a new horizon in disease diagnosis due to its ability to detect H2O2 which is a potential biomarker for many diseases. Our work is appealing to researchers working in nanotechnology, and the development of new bioassays as well as POC devices.

A Novel Electrochemical Aptasensor Based on a New Platform of Samarium Molybdate Flower-like Nanoparticles@Poly(pyrrole) for Non-Invasive Determination of Saliva CORTISOL.

Cortisol, a famous stress biomarker, can be considered a potential predictor of cardiac diseases in humans. The presence of cortisol in saliva has encouraged researchers to design point-of-care devices for cortisol concentration in biological fluids. Here, human salivary cortisol was analyzed through a new non-invasive voltammetric aptasensor. Although cortisol is an electroactive compound, generally, the reduction in the current peak has been considered; however, this does not show a strong signal on a bare electrode surface, especially at low concentration levels. Hence, in this study, cortisol concentration was measured electrochemically and indirectly by monitoring the difference between electrochemical probe signals in the presence and absence of cortisol. A new polymeric nanocomposite of samarium molybdate flower-like nanoparticles decorated in poly(pyrrole) was electro-synthesized on the surface of a glassy carbon electrode. Then, reduced graphene oxide was cast on the surface. Finally, the cortisol aptamer was immobilized covalently on the reduced graphene oxide. This platform was used to increase the oxidation current peak of the ferricyanide solution as a probe as well as its electrocatalyst. The novel designed polymeric has the potential ability for effective immobilization of aptamers on the electrode surface without decreasing their biological activities. Additionally, it can enhance the probe electrochemical signal. The differential pulse voltammetric method (DPV) was applied as the detection technique. By optimizing the effective parameters, a determination range of 5.0 × 10−14–1.5 × 10−11 mol/L and a limit of detection of 4.5 × 10−14 mol/L were obtained. Selectivity of the proposed aptasensor relative to β-estradiol, progesterone and also prednisolone was studied as well. Finally, cortisol in a healthy human saliva sample was successfully analyzed by the proposed biosensors.

Turn-on electrochemiluminescence sensing of melatonin based on graphitic carbon nitride nanosheets

• A sensitive turn-on ECL sensor was designed for detection of melatonin. • Graphitic carbon nitride nano-sheets (g-C3N4 NSs) were used as an ECL probe. • The developed ECL sensor exhibited low detection limit of 6.2 × 10-15 M. • The ECL sensor was successfully applied to detect melatonin in human saliva samples. In this work, a turn-on electrochemiluminescence (ECL) sensor for sensitive quantification of melatonin based on graphite carbon nitride (g-C 3 N 4 ) was prepared on the surface of a glassy carbon electrode (GCE). In this ECL sensor, graphitic carbon nitride nano-sheets (g-C 3 N 4 NSs) were used as an ECL probe. The ECL intensity of the sensor was gradually increased with the addition of melatonin concentration. The possible mechanism for enhanced ECL signal of g-C 3 N 4 by melatonin is investigated and discussed in detail. Under optimal conditions, a novel, sensitive and selective ECL sensor has been constructed for the detection of melatonin, with a wide linear response range from 1.0 × 10 -14 to 1.0 × 10 -9 M, and an excellent detection limit of 6.2 × 10 -15 M (6.2 fM). Furthermore, the proposed sensor was successfully applied to detect melatonin in human saliva samples with excellent precision, remarkable selectivity, and long-lasting stability.

All-in-one calcium nanoflowers for dual outputs biosensor: A simultaneous strategy for depression drug evaluation and non-invasive stress assessment

Although enzyme-based signal amplification has been well developed for biosensors, their application in low-abundance biomarker under complicated conditions detection remains challenge. Cortisol is a steroid hormone and a quantitative evaluation of cortisol can objectively assess stress and depression. However, various factors can induce slight cortisol changes in body fluids, and this in turn sets a strict requirement for bedside testing of cortisol for evaluation of stress. Herein, all-in-one calcium nanoflowers (CaHPO4-AM-HRP-SA NFs) integrated with horseradish peroxidase (HRP), α-amylase (α-AM), and streptavidin (SA) have been synthesized to develop a simple but powerful biosensor for cortisol detection. High specific surface area and allosteric modulator provided by the hybrid nanoflowers as inherent advantages significantly boosted the catalytical ability and stability compared with the free enzymes. CaHPO4-AM-HRP-SA NFs also endowed the sensor with two output signals of one sample, leading the as-prepared sensor to realize self-calibration detection. Aside from using a traditional microplate reader to measure the signal, it could also be read out by a handheld blood glucose meter and a mobile phone. The sensor exhibited attractive simplicity and sensitivity with a low LOD of 98.5 pg mL−1. It accomplished the sensitive evaluation of cortisol in rat serum and assessed the antidepressant effects of different medications. The non-invasive and reliable cortisol detection is also achieved in human urine and saliva samples. Overall, we have demonstrated that the sensor can be deployed as a promising platform to evaluate drug efficiency and monitor stress in a simple and non-invasive manner.

Distance-Based All-In-One Immunodevice for Point-of-Care Monitoring of Cytokine Interleukin-6.

We report the development of a distance-based paper analytical device combined with a hydrophilic bridge valve (B-dPAD) as a quantitative immunoassay method to monitor human interleukin-6 (IL-6) in human samples. Our device design features (i) a circular sample inlet zone, (ii) a circular capture zone with immobilized anti-IL-6 (anti-Ab1), and (iii) a detection zone channel coated with methylene blue (MB). Two hydrophilic valves are positioned between these three zones. IL-6 levels were determined quantitatively by measuring the extent of degradation of MB to a colorless product along the length of the detection zone channel. Following method optimization, we obtained a linear range from 0.05 to 25.0 pg/mL (R2 = 0.9995) and a detection limit (LOD) of 0.05 pg/mL by the naked-eye readout. This is directly within the clinically relevant range. The system does not require any external instrumentation, and the bridge valves can be easily connected and disconnected by a minimally trained operator. The total analysis time is 35 min, significantly reduced from a typical ELISA assay, which takes around 1 h since the B-dPAD workflow circumvents washing steps. The device was tested for IL-6 quantification in human saliva and urine samples of volunteers, with no significant difference found between our method and the standard clinical laboratory method at 95% confidence levels. Recoveries ranged from 98 to 105% with the highest standard deviation at 3.9%. Our B-dPAD immunodevice is therefore a promising approach for rapid IL-6 monitoring in the context of point-of-care diagnostics and analysis in resource-limited settings.

Machine Learning-Based Quantification of (-)-trans-Δ-Tetrahydrocannabinol from Human Saliva Samples on a Smartphone-Based Paper Microfluidic Platform.

(−)-trans-Δ-Tetrahydrocannabinol (THC) is a major psychoactive component in cannabis. Despite the recent trends of THC legalization for medical or recreational use in some areas, many THC-driven impairments have been verified. Therefore, convenient, sensitive, quantitative detection of THC is highly needed to improve its regulation and legalization. We demonstrated a biosensor platform to detect and quantify THC with a paper microfluidic chip and a handheld smartphone-based fluorescence microscope. Microfluidic competitive immunoassay was applied with anti-THC-conjugated fluorescent nanoparticles. The smartphone-based fluorescence microscope counted the fluorescent nanoparticles in the test zone, achieving a 1 pg/mL limit of detection from human saliva samples. Specificity experiments were conducted with cannabidiol (CBD) and various mixtures of THC and CBD. No cross-reactivity to CBD was found. Machine learning techniques were also used to quantify the THC concentrations from multiple saliva samples. Multidimensional data were collected by diluting the saliva samples with saline at four different dilutions. A training database was established to estimate the THC concentration from multiple saliva samples, eliminating the sample-to-sample variations. The classification algorithms included k-nearest neighbor (k-NN), decision tree, and support vector machine (SVM), and the SVM showed the best accuracy of 88% in estimating six different THC concentrations. Additional validation experiments were conducted using independent validation sample sets, successfully identifying positive samples at 100% accuracy and quantifying the THC concentration at 80% accuracy. The platform provided a quick, low-cost, sensitive, and quantitative point-of-care saliva test for cannabis.

Direct decoration of carbon nanohorns with binary nickel-cobalt sulfide nanosheets towards non-enzymatic glucose sensing in human fluids

A highly controllable, green, and rapid strategy is demonstrated for fabricating of highly sensitive non-enzymatic glucose sensing platforms. Carbon nanohorns (CNHs) were decorated onto the screen-printed electrodes. Binary nickel-cobalt sulfide (NiCo-S) nanosheets (NSs) were then deposited on CNH-casted electrodes by a facile and scalable method. Following detailed structural characterization and the electrocatalytic activity of the fabricated NiCo-S/CNH electrodes towards electro-oxidation of glucose was examined in detail. The proposed electrodes operated within two distinct linear dynamic ranges of 0.001- 0.330 mM and 0.330 - 4.53 mM with sensitivities of 1842 µA.mM−1.cm−2 and 854 µA.mM−1.cm−2, respectively. Analytical sensitivity of 467 µA.mM−1.cm−2, within the linear dynamic range of 0.5 ∼ 6 mM, was obtained for the static chronoamperometric measurements. The proposed sensor showed outstanding selectivity against isostructures and co-existing interferences, long-term stability, fast response (1.7 s), excellent poisoning resistance against chloride ions, good repeatability, and high reproducibility. The structural and electrochemical properties of the NiCo-S/CNHs were also compared to its mono-metallic counterparts. Finally, to demonstrate the capability of the fabricated electrodes for glucose measurements in physiological specimens, the glucose level was successfully measured in human blood serum, urine, and saliva samples, without any sample pre-treatment.

Fabric Phase Sorptive Extraction (FPSE) as an efficient sample preparation platform for the extraction of antidepressant drugs from biological fluids

• Fast method for simultaneous analysis of seven ADs. • Analysis of biological matrices. • FPSE application in conventional and unconventional matrices. • Method suitable for TDM of ADs. • Performances comparable to a more complex and expensive instrumentations. The quantification and interpretation of drug concentrations in biological matrices to optimize pharmacotherapy and to perform the therapeutic drug monitoring (TDM) is particularly important for compounds with narrow therapeutic ranges, known to cause adverse effects. In these cases, the biomonitoring is essential to avoid the toxicity and side effects. In this study, an innovative Fabric Phase Sorptive Extraction (FPSE) followed by high performance liquid chromatography-photodiode array detection (FPSE–HPLC–PDA) method was optimized and validated for the extraction and quantitative evaluation of seven antidepressant drugs (ADs, venlafaxine, citalopram, paroxetine, fluoxetine, sertraline, amitriptyline, and clomipramine) in human whole blood, urine, and saliva samples. The best chromatographic separation was obtained using a reverse phase column and ammonium acetate (50 mM, pH 5.5) and acetonitrile (AcN) as mobile phases, with 0.3% of triethylamine (TEA) for the best peak shape. The used sample preparation technique, FPSE, developed in 2014, has offered numerous advantages such as low consumption of organic solvents, no sample pretreatment, and reduced overall sample preparation time. Among all tested membranes, sol-gel carbowax (CW 20 M) sorbent, coated on cellulose FPSE media, was the most efficient. The developed method provides satisfactory limit of detection of 0.06 μg/mL for all analytes except for venlafaxine that was 0.04 μg/mL. Both RSD% and BIAS% gave values below ±15%, according to current guidelines. Finally, real samples analyzes were carried out, comparing the obtained data with the anamnestic data of the subjects, confirmed the validity of the method.

A Bioinformatics Approach to MicroRNA-Sequencing Analysis Based on Human Saliva Samples of Patients with Endometriosis.

Endometriosis, defined by the presence of endometrium-like tissue outside the uterus, affects 2–10% of the female population, i.e., around 190 million women, worldwide. The aim of the prospective ENDO-miRNA study was to develop a bioinformatics approach for microRNA-sequencing analysis of 200 saliva samples for miRNAome expression and to test its diagnostic accuracy for endometriosis. Among the 200 patients, 76.5% (n = 153) had confirmed endometriosis and 23.5% (n = 47) had no endometriosis (controls). Small RNA-seq of 200 saliva samples yielded ~4642 M raw sequencing reads (from ~13.7 M to ~39.3 M reads/sample). The number of expressed miRNAs ranged from 1250 (outlier) to 2561 per sample. Some 2561 miRNAs were found to be differentially expressed in the saliva samples of patients with endometriosis compared with the control patients. Among these, 1.17% (n = 30) were up- or downregulated. Among these, the F1-score, sensitivity, specificity, and AUC ranged from 11–86.8%, 5.8–97.4%, 10.6–100%, and 39.3–69.2%, respectively. Here, we report a bioinformatic approach to saliva miRNA sequencing and analysis. We underline the advantages of using saliva over blood in terms of ease of collection, reproducibility, stability, safety, non-invasiveness. This report describes the whole saliva transcriptome to make miRNA quantification a validated, standardized, and reliable technique for routine use. The methodology could be applied to build a saliva signature of endometriosis.

Hydrophobic deep eutectic solvent‐based microextraction method for the simultaneous extraction of two benzodiazepines from saliva samples before determination by 96‐well microplates‐based spectrophotometer with the aid of chemometrics

AbstractIn this study, a 96‐well microplates‐based spectrophotometer with the aid of the partial least squares (PLS) approach was used for the simultaneous determination of more common benzodiazepines, clonazepam, and nitrazepam, in human saliva samples. For this purpose, a hydrophobic deep eutectic solvent (DES) based on choline chloride and 1‐phenylethanol in the mole ratio of 1:4 was prepared and utilized in the ultrasound‐assisted emulsification‐microextraction (USAEME) method followed by back‐extraction to retrieve the drugs from the DES phase to 200 μL of 0.1 M NaOH solution. Central composite design (CCD) in combination with the desirability function was used for multi‐response optimization of different parameters and the optimum values were found to be: pH 2.06; DES volume, 247.1 μL; NaCl, 1.55% (w/v); sonication time, 5.33 min. The DES‐based USAEME method showed wide linear ranges of 0.1–6 and 0.2‐8 μg/mL and good limits of detection of 0.02 and 0.05 μg/mL for clonazepam and nitrazepam, respectively. Furthermore, the method was successfully used for determining the drugs in human saliva samples.