Artificial Human Saliva Research Articles

Electrochemical Sensor for Levetiracetam Therapeutic Monitoring in Biological Fluid Utilizing Molecular Imprinted Polymer Electrografted onto Pencil Graphite Electrode

A newly-developed, molecularly imprinted copolymer (MIP) has been constructed and effectively employed as a highly-selective recognition element for anti-epileptic drug; levetiracetam (LEV) in low concentrations. The MIP sensor for LEV was fabricated via in situ electrochemical co-polymerization of o-phenylenediamine (o-PD) and L-dopa as functional monomers in the presence of LEV as a template molecule onto pencil graphite electrode. UV-Spectrophotometric technique was carried out to inspect and assess the template-monomer binding interactions. The molecularly imprinted copolymer’s structure and morphology were examined through X-ray photoelectron spectroscopy and scanning electron microscopy. Multiple experimental parameters have been investigated and optimized in order to improve the sensor’s responsiveness towards LEV. Utilizing differential pulse voltammetry, quantitative measurements of MIP-based LEV detection were achieved indirectly by measuring the decrease in response of redox probe upon binding of LEV to the 3D cavities of MIP in phosphate buffer saline pH 7.40. The method’s validation was performed in accordance with ICH guidelines. The fabricated sensor showed a linear voltammetric response with a linearity range of (1.5 × 10–12–1.0 × 10–10 M). The sensor was able to effectively detect LEV in spiked artificial human saliva.

Electrochemical Multiplexed N-Terminal Natriuretic Peptide and Cortisol Detection in Human Artificial Saliva: Heart Failure Biomedical Application

The early detection at low concentration, by non-invasive methods, of cardiac biomarkers in physiological fluids has attracted the interest of researchers over the last decade. This enables early diagnosis and prediction of the first signs of heart failure (HF). In this respect, the analysis of human saliva remains the most suitable medium for this non-invasive approach, as it contains a highly interesting biological matrix for general health and disease monitoring. In this work, we developed a highly sensitive multiplexed immunosensor for direct simultaneous detection of both N-terminal Natriuretic Peptide (NT-proBNP) and Cortisol in human artificial saliva (AS). The developed biosensor platform based on silicon nitride substrate was composed from four gold working microelectrodes (WEs) and an integrated counter and reference microelectrode. Gold WEs were biofunctionalized through carboxyl diazonium (4-APA) to immobilize both anti-NT-proBNP and anti-Cortisol antibodies for simultaneous detection. The electroaddressing of the 4-APA onto the gold WE surfaces was realized with cyclic voltammetry (CV), while the interaction between antibodies and antigens in PBS was monitored using electrochemical impedance spectroscopy (EIS). The antigen detection in human AS was realized with EIS combined with the standard addition method. The immunosensor was highly sensitive and selective toward the corresponding biomarkers in both PBS and artificial human saliva as well as in the presence of other potential interfering biomarkers such as tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10). The limit of detection (LOD) was at 0.2 pg/mL for NT-proBNP within the range of 0.03 to 0.9 pg/mL, while the LOD for Cortisol was 0.06 ng/mL within the range of 0.02 to 0.6 ng/mL for Cortisol in artificial saliva. The developed immunosensor is very promising for significant detection in physiological media, and time reducing as it allows the simultaneous detection of various biomarkers.

Viricidal Efficacy of a 405-nm Environmental Decontamination System for Inactivation of Bacteriophage Phi6: Surrogate for SARS-CoV-2.

The highly transmittable nature of SARS-CoV-2 has increased the necessity for novel strategies to safely decontaminate public areas. This study investigates the efficacy of a low irradiance 405-nm light environmental decontamination system for the inactivation of bacteriophage phi6 as a surrogate for SARS-CoV-2. Bacteriophage phi6 was exposed to increasing doses of low irradiance (~0.5 mW cm-2 ) 405-nm light while suspended in SM buffer and artificial human saliva at low (~103-4 PFU mL-1 ) and high (~107-8 PFU mL-1 ) seeding densities, to determine system efficacy for SARS-CoV-2 inactivation and establish the influence of biologically relevant suspension media on viral susceptibility. Complete/near-complete (≥99.4%) inactivation was demonstrated in all cases, with significantly enhanced reductions observed in biologically relevant media (P < 0.05). Doses of 43.2 and 172.8 J cm-2 were required to achieve ~3 log10 reductions at low density, and 97.2 and 259.2 J cm-2 achieved ~6 log10 reductions at high density, in saliva and SM buffer, respectively: 2.6-4 times less dose was required when suspended in saliva compared to SM buffer. Comparative exposure to higher irradiance (~50 mW cm-2 ) 405-nm light indicated that, on a per unit dose basis, 0.5 mW cm-2 treatments were capable of achieving up to 5.8 greater log10 reductions with up to 28-fold greater germicidal efficiency than that of 50 mW cm-2 treatments. These findings establish the efficacy of low irradiance 405-nm light systems for inactivation of a SARS-CoV-2 surrogate and demonstrate the significant enhancement in susceptibility when suspended in saliva, which is a major vector in COVID-19 transmission.

Characterization of Metallic Off-Flavors in Drinking Water: Health, Consumption, and Sensory Perception.

Characterization of taste- and flavor-producing metals, namely iron and copper, in drinking water is a multifaceted subject. Both metals are essential nutrients, can be toxic, and are known to produce unpleasant tastes and flavor sensations in drinking water. Ingestion of trace metal contaminants through drinking water is a probable source of human exposure. Biochemical mechanisms of metallic flavor perception have been previously described; however, less is known about how variations in salivary constituents might impact individuals' sensitivities to metallic flavors and beverage consumption behaviors. This research presents findings from in vitro experiments, using artificial human saliva, to better understand the role of salivary lipids and proteins on metallic flavor production as measured by biomarkers of metal-induced oxidative stress. The results indicate that metal-induced lipid oxidation, as measured by thiobarbituric acid reactive substances (TBARS), is dominated by salivary proteins, is slightly inhibited in the presence of salivary nitrite, and is detectable by the TBARS method at and above respective concentrations of 9 µM (0.5 mg/L) and 90 µM (5 mg/L), which are both above the aesthetic standards for iron (0.3 mg/L) and copper (1.0 mg/L) in drinking water. Preliminary study with human subjects indicated that reduction in metallic flavor sensitivity, as measured by the best estimate flavor threshold for ferrous iron among 33 healthy adults aged 19-84 years old (22 females), corresponded with reduced drinking water consumption and increased caloric beverage intake among older subjects (>60 years), as determined by a validated self-reported beverage intake questionnaire. These findings provide insights for further research to examine how salivary constituents can impact humans' sensory abilities in detecting metallic off-flavors in water, and how reduced metallic flavor sensitivity may influence beverage choices and drinking water consumption.

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.

Exploration of Basella alba mucilage as a novel adjuvant in Pharmaceutical Formulation

Basella alba is a commonly available plant having significant quantity of mucilage present in its leaves and stem. The review focuses on the method of extraction of mucilage, its chemical composition, monosaccharide composition and various chemical and physical properties of mucilage. The rheological properties of B.alba mucilage, exhibiting pseudoplastic flow enable it to be used for formulating artificial human saliva. The B.alba mucilage is found to be used as disintegrating agent, binding agent, suspending agent, matrix former, gelling agent, encapsulating agent and as release retardant in pharmaceutical formulation. The properties of mucilage have been evaluated against standard excipient and confirmed of their adjuvant properties. The mucilage also possesses drag reducing ability due to which it can be used as natural flow improver in pipelines for reducing the frictional pressure drop. The plant is rapidly growing, commonly available and possesses wide additive properties; even then it remains under- utilized as pharmaceutical excipient so its versatility in targeted drug delivery, micro-/nano-drug delivery, tissue engineering and stimuli responsive delivery system is highly desirable. Apart from this, several opportunities of mucilage which are left unexplored is discussed in the review like formation of BioMEMS, formation of microneedles and as theranostic agents. Thus, the review explores various pharmaceutical applications and opportunities of B.alba mucilage to act as smart novel carrier in pharmaceutical formulation.

Controlled, partially exfoliated, self-supported functionalized flexible graphitic carbon foil for ultrasensitive detection of SARS-CoV-2 spike protein

This paper reports on an ultrasensitive and label-free electrochemical immunosensor for monitoring the SARS-CoV-2 spike protein (SARS-CoV-2 SP). A self-supported electrode, which can simultaneously serve as an antibody immobilization matrix and electron transport channel, was initially fabricated by a controlled partial exfoliation of a flexible graphitic carbon foil (GCF). Mild acidic treatment enabled the partial oxidation and exfoliation (down to a few layers) of the flexible GCF; this also provided a high percentage of oxygen functionality and an enhanced surface roughness. The substrate electrode was further functionalized with ethylenediamine (EDA) to provide a suitable platform with even a higher surface roughness, for the covalent immobilization of an anti-SARS-CoV-2 antibody. The change in the current response for the [Fe(CN)6]3−/4− redox couple, induced by the binding of SARS-CoV-2 SP to the antibody immobilized on the electrode surface, was used to determine the SARS-CoV-2 SP concentration. The immunosensor thus prepared could detect SARS-CoV-2 SP within 30 min with high reproducibility and specificity over a wide concentration range (0.2–100 ng/mL). Detection limits of 25 pg/mL and 27 pg/mL were found in a phosphate buffer solution (pH 7.4), and diluted blood plasma, respectively. The immunosensor was also employed to detect SARS-CoV-2 SP in artificial human saliva.

Rapid Fabrication of Superhydrophobic Virtual Walls for Microfluidic Gas Extraction and Sensing.

Based on the virtual walls concept, where fluids are guided by wettability, we demonstrate the application of a gas phase extraction microfluidic chip. Unlike in previous work, the chip is prepared using a simple, rapid, and low-cost fabrication method. Channels were cut into double-sided adhesive tape (280 µm thick) and bonded to hydrophilic glass slides. The tape was selectively made superhydrophobic by ‘dusting’ with hydrophobic silica gel to enhance the wettability contrast at the virtual walls. Finally, the two glass slides were bonded using tape, which acts as a spacer for gas transport from/to the guided liquids. In our example, the virtual walls create a stable liquid–vapor–liquid flow configuration for the extraction of a volatile analyte (ammonia), from one liquid stream to the other through the intermediate vapor phase. The collector stream contained a pH indicator to visualize the mass transport. Quantitative analysis of ammonium hydroxide in the sample stream (<1 mM) was possible using a characteristic onset time, where the first pH change in the collector stream was detected. The effect of gap length, flow rates, and pH of the collector stream on the onset time is demonstrated. Finally, we demonstrate the analysis of ammonium hydroxide in artificial human saliva to show that the virtual walls chip is suitable for extracting volatile analytes from biofluids.

On-chip absorption spectroscopy enabled by graded index fiber tips.

This paper describes the design and characterization of miniaturized optofluidic devices for sensing based on integrating collimating optical fibers with custom microfluidic chips. The use of collimating graded-index fiber (GIF) tips allows for effective fiber-channel-fiber interfaces to be realized when compared with using highly-divergent standard single-mode fiber (SMF). The reduction in both beam divergence and insertion losses for the GIF configuration compared with SMF was characterized for a 10.0 mm channel. Absorption spectroscopy was demonstrated on chip for the measurement of red color dye (Ponceau 4R), and the detection of thiocyanate in water and artificial human saliva. The proposed optofluidic setup allows for absorption spectroscopy measurements to be performed with only 200 µL of solution which is an order of magnitude smaller than for standard cuvettes but provides a comparable sensitivity. The approach could be integrated into a lab-on-a-chip system that is compact and does not require free-space optics to perform absorption spectroscopy.

Metals and Metalloids Release from Orthodontic Elastomeric and Stainless Steel Ligatures: In Vitro Risk Assessment of Human Exposure

Elastomeric ligatures are increasingly used as a part of esthetic orthodontic treatment, particularly in children. The aim of the present study was to experimentally test whether these appliances may contribute to exposure to toxic elements. In the present study, elastomeric ligatures (ELs) were incubated in artificial human saliva for 1 month (a typical period of their use) and the release of 21 metals (Ba, Cd, Co, Cr, Cu, Fe, Li, Mn, Mg, Mo, Ni, Pb, Rb, Tl, Ti, Sb, Sr, Sn, Zn, U, V) and 2 metalloids (As and Ge) was studied using inductively coupled plasma–mass spectrometry. For comparison, stainless steel ligatures (SLs) were incubated for 1, 3, and 6 months (since sometimes their use is prolonged) under similar conditions. The determined metal levels were compared to the corresponding safety limits for human exposure. During 1 month, the ELs released Cd, Co, Cr, Mn, Ni, and Sn at total mean ± SD level of 0.31 ± 0.09, 0.98 ± 0.30, 3.96 ± 1.31, 14.7 ± 8.5, 13.8 ± 4.8, and 49.5 ± 27.7 μg, respectively. Other elements were always below the detection limits. In case of SL, the release of Co, Cr, Fe, Ni, Mn, and Sn was observed, and the determined values increased over the studied period. After 6 months, their total mean ± SD levels amounted to 28.6 ± 0.2, 21.7 ± 0.2, 623.5 ± 3.0, 1152.7 ± 1.8, 5.5 ± 0.3, and 22.6 ± 0.2 μg, respectively. The released metal levels from both ligature types were always below safety limits. The release of Ni from SL during 6 months would constitute 5.0 and 11.5% of tolerable intake in adults and children, respectively. The results of this in vitro study highlight that the use of ligatures in orthodontic treatment can be considered safe in terms of metal exposure although elastic ligatures replaced on a monthly basis appear to be advantageous in comparison to the prolonged use of stainless steel appliances.

Capacitance electrochemical biosensor based on silicon nitride transducer for TNF-α cytokine detection in artificial human saliva: Heart failure (HF)

In the present study, we have developed a capacitance electrochemical biosensor based on silicon nitride substrate (Si3N4/SiO2/Si[P]/Al) for Tumour Necrosis Factor Alpha (TNF-α) cytokines detection. Micro-contact printing, Fluorescence microscopy characterization and contact angle measurement (CAM) were carried out during the bio-functionalization of the biosensor surface. Mott-Schottky analyses were applied for TNF-α detection within the range of 1 pg/mL to 30 pg/mL in which the immunosensor has exhibited a good linearity, a sensitivity of 4 mV.pM−1 and 4.4 mV.pM−1 in PBS and artificial saliva (AS) respectively. While the LOD was found at 0.38 pg/mL and 1 pg/mL in PBS and AS respectively. The developed immunosensor has also demonstrated a high and good selectivity for TNF-α detection in human AS when compared to other interferences like Cortisol and Interleukin-10. The performances of the developed biosensor are very promising for biomedical application to predict the first sign of inflammation.

Salivary Detection of Dengue Virus NS1 Protein with a Label-Free Immunosensor for Early Dengue Diagnosis.

Dengue virus (DENV) is a highly pathogenic, arthropod-borne virus transmitted between people by Aedes mosquitoes. Despite efforts to prevent global spread, the potential for DENV epidemics is increasing world-wide. Annually, 3.6 billion people are at risk of infection. With no licensed vaccine, early diagnosis of dengue infection is critical for clinical management and patient survival. Detection of DENV non-structural protein 1 (NS1) is a clinically accepted biomarker for the early detection of DENV infection. Unfortunately, virtually all of the laboratory and commercial DENV NS1 diagnostic methods require a blood draw for sample analysis, limiting point-of-care diagnostics and decreases patient willingness. Alternatively, NS1 in human saliva has been identified for the potential early diagnosis of DENV infection. The collection of saliva is simple, non-invasive, painless, and inexpensive, even by minimally trained personnel. In this study, we present a label-free chemiresistive immunosensor for the detection of the DENV NS1 protein utilizing a network of single-walled carbon nanotubes functionalized with anti-dengue NS1 monoclonal antibodies. NS1 was successfully detected in adulterated artificial human saliva over the range of clinically relevant concentrations with high sensitivity and selectivity. It has potential application in clinical diagnosis and the ease of collection allows for self-testing, even within the home.

Tribocorrosion mechanisms of Ti6Al4V biomedical alloys in artificial saliva with different pHs

Titanium and its alloys has been widely used for the design of dental implants because of its biocompatibility, mechanical properties and corrosion resistance. The powder-metallurgy process is a promising alternative to the casting fabrication process of titanium alloys for bone implants design as the porous structure mimics the natural bone structures, allowing the bone to grow into the pores which results in a better fixation of the artificial implant. However, under in vivo conditions the implants are subjected to tribocorrosion phenomenon, which consists in the degradation mechanisms due to the combined effect of wear and corrosion. The aim of this study is to evaluate the tribocorrosion behaviour of cast and sintered Ti6Al4V biomedical alloy for dental applications using the cast material as reference. Titanium samples were tested in artificial human saliva solution with three different pHs (3, 6, 9) and in an acidic saliva with 1000 ppm fluorides (AS-3-1000F−) by different electrochemical techniques (potentiodynamic curves, potentiostatic tests and tribo-electrochemical tests). Cast and sintered titanium alloys exhibit the same tribocorrosion mechanisms in AS independently of the pH which consists in plastic deformation with passive dissolution, but the addition of fluorides to the acidified solution changes the degradation mechanism towards active dissolution of the titanium alloys.

Influence of the fabrication process and fluoride content on the tribocorrosion behaviour of Ti6Al4V biomedical alloy in artificial saliva

Titanium and its alloys are widely used as dental implants due to their low density, excellent biocompatibility, mechanical properties and corrosion resistance. However, during their lifetime Ti6Al4V biomedical alloys are subjected to different mechanical actions (i.e., sliding or fretting), thus resulting in a tribocorrosion system, which is an irreversible material degradation process due to the combined effect of corrosion and wear. In this study, the tribo-electrochemical behavior of cast and sintered (by powder metallurgy) Ti6Al4V alloy in artificial human saliva solution without and with fluoride additions of 100ppm and 1000ppm and in an average industrial mouth wash solution with a 112ppm fluoride content have been investigated by different electrochemical techniques. The same tribocorrosion mechanisms were found in the cast and sintered titanium alloys, although slightly different wear debris behavior was observed. At low applied passive potentials, wear rates are similar to those obtained under equilibrium conditions (Open Circuit Potential). There exists a critical fluoride concentration above which corrosion and tribocorrosion rates increase.