Flexible organic electrochemical transistors (OECTs) are successfully used as high‐performance enzyme biosensors, such as uric acid (UA) and cholesterol sensors. The sensitivity and selectivity of the sensors can be simultaneously enhanced by co‐modifying the gate electrodes with positively/negatively charged bilayer polymer films and enzymes. These OECT‐based UA sensors are successfully utilized for non‐invasive UA detection in human saliva.
The mobile health market is rapidly expanding and portable diagnostics tools offer an opportunity to decrease costs and increase the availability of healthcare. Here we present a smartphone based accessory and method for the rapid colorimetric detection of pH in sweat and saliva. Sweat pH can be correlated to sodium concentration and sweat rate in order to indicate to users the proper time to hydrate during physical exercise and avoid the risk of muscle cramps. Salivary pH below a critical threshold is correlated with enamel decalcification, an acidic breakdown of calcium in the teeth. We conduct a number of human trials with the device on a treadmill to demonstrate the ability to monitor changes in sweat pH due to exercise and electrolyte intake and predict optimal hydration. Additionally, we perform trials to measure salivary pH over time to monitor the effects of diet on oral health risks.
There is an increasing interest in using microRNAs (miRNA) as biomarkers in autoimmune diseases. They are easily accessible in many body fluids but it is controversial if they are circulating freely or are encapsulated in microvesicles, particularly exosomes. We investigated if the majority of miRNas in serum and saliva are free-circulating or concentrated in exosomes. Exosomes were isolated by ultracentrifugation from fresh and frozen human serum and saliva. The amount of selected miRNAs extracted from the exosomal pellet and the exosome-depleted serum and saliva was compared by quantitative RT-PCR. Some miRNAs tested are ubiquitously expressed, others were previously reported as biomarkers. We included miRNAs previously reported to be free circulating and some thought to be exosome specific. The purity of exosome fraction was confirmed by electronmicroscopy and western blot. The concentration of miRNAs was consistently higher in the exosome pellet compared to the exosome-depleted supernatant. We obtained the same results using an equal volume or equal amount of total RNA as input of the RT-qPCR. The concentration of miRNA in whole, unfractionated serum, was between the exosomal pellet and the exosome-depleted supernatant. Selected miRNAs, which were detectable in exosomes, were undetectable in whole serum and the exosome-depleted supernantant. Exosome isolation improves the sensitivity of miRNA amplification from human biologic fluids. Exosomal miRNA should be the starting point for early biomarker studies to reduce the probability of false negative results involving low abundance miRNAs that may be missed by using unfractionated serum or saliva.
In this review, we provide an update on the current and future applications of saliva for diagnostic purposes. There are many advantages of using saliva as a biofluid. Its collection is fast, easy, inexpensive, and non-invasive. In addition, saliva, as a “mirror of the body,” can reflect the physiological and pathological state of the body. Therefore, it serves as a diagnostic and monitoring tool in many fields of science such as medicine, dentistry, and pharmacotherapy. Introduced in 2008, the term “Salivaomics” aimed to highlight the rapid development of knowledge about various “omics” constituents of saliva, including: proteome, transcriptome, micro-RNA, metabolome, and microbiome. In the last few years, researchers have developed new technologies and validated a wide range of salivary biomarkers that will soon make the use of saliva a clinical reality. However, a great need still exists for convenient and accurate point-of-care devices that can serve as a non-invasive diagnostic tool. In addition, there is an urgent need to decipher the scientific rationale and mechanisms that convey systemic diseases to saliva. Another promising technology called liquid biopsy enables detection of circulating tumor cells (CTCs) and fragments of tumor DNA in saliva, thus enabling non-invasive early detection of various cancers. The newly developed technology—electric field-induced release and measurement (EFIRM) provides near perfect detection of actionable mutations in lung cancer patients. These recent advances widened the salivary diagnostic approach from the oral cavity to the whole physiological system, and thus point towards a promising future of salivary diagnostics for personalized individual medicine applications including clinical decisions and post-treatment outcome predictions. Impact statement The purpose of this mini-review is to make an update about the present and future applications of saliva as a diagnostic biofluid in many fields of science such as dentistry, medicine and pharmacotherapy. Using saliva as a fluid for diagnostic purposes would be a huge breakthrough for both patients and healthcare providers since saliva collection is easy, non-invasive and inexpensive. We will go through the current main diagnostic applications of saliva, and provide a highlight on the emerging, newly developing technologies and tools for cancer screening, detection and monitoring.
BACKGROUND: Over the past 10 years, the use of saliva as a diagnostic fluid has gained attention and has become a translational research success story. Some of the current nanotechnologies have been demonstrated to have the analytical sensitivity required for the use of saliva as a diagnostic medium to detect and predict disease progression. However, these technologies have not yet been integrated into current clinical practice and work flow. CONTENT: As a diagnostic fluid, saliva offers advantages over serum because it can be collected noninvasively by individuals with modest training, and it offers a cost-effective approach for the screening of large populations. Gland-specific saliva can also be used for diagnosis of pathology specific to one of the major salivary glands. There is minimal risk of contracting infections during saliva collection, and saliva can be used in clinically challenging situations, such as obtaining samples from children or handicapped or anxious patients, in whom blood sampling could be a difficult act to perform. In this review we highlight the production of and secretion of saliva, the salivary proteome, transportation of biomolecules from blood capillaries to salivary glands, and the diagnostic potential of saliva for use in detection of cardiovascular disease and oral and breast cancers. We also highlight the barriers to application of saliva testing and its advancement in clinical settings. SUMMARY: Saliva has the potential to become a first-line diagnostic sample of choice owing to the advancements in detection technologies coupled with combinations of biomolecules with clinical relevance. (C) 2011 American Association for Clinical Chemistry
BACKGROUND: Extracellular RNAs (exRNAs) in human body fluids are emerging as effective biomarkers for detection of diseases. Saliva, as the most accessible and non-invasive body fluid, has been shown to harbor exRNA biomarkers for several human diseases. However, the entire spectrum of exRNA from saliva has not been fully characterized. METHODS: Using high-throughput RNA sequencing (RNA-Seq), we conducted an in-depth bioinformatic analysis of noncoding RNAs (ncRNAs) in human cell-free saliva (CFS) from healthy individuals, with a focus on microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and circular RNAs (circRNAs). RESULTS: Our data demonstrated robust reproducibility of miRNA and piRNA profiles across individuals. Furthermore, individual variability of these salivary RNA species was highly similar to those in other body fluids or cellular samples, despite the direct exposure of saliva to environmental impacts. By comparative analysis of >90 RNA-Seq data sets of different origins, we observed that piRNAs were surprisingly abundant in CFS compared with other body fluid or intracellular samples, with expression levels in CFS comparable to those found in embryonic stem cells and skin cells. Conversely, miRNA expression profiles in CFS were highly similar to those in serum and cerebrospinal fluid. Using a customized bioinformatics method, we identified >400 circRNAs in CFS. These data represent the first global characterization and experimental validation of circRNAs in any type of extracellular body fluid. CONCLUSIONS: Our study provides a comprehensive landscape of ncRNA species in human saliva that will facilitate further biomarker discoveries and lay a foundation for future studies related to ncRNAs in human saliva. (C) 2014 American Association for Clinical Chemistry
Dual screen-printed carbon electrodes modified with 4-carboxyphenyl-functionalized double-walled carbon nanotubes (HOOC-Phe-DWCNTs/SPCEs) have been used as scaffolds for the preparation of electrochemical immunosensors for the simultaneous determination of the cytokines Interleukin-1β (IL-1β) and factor necrosis tumor α (TNF-α). IL-1β. Capture antibodies were immobilized onto HOOC-Phe-DWCNTs/SPCEs in an oriented form making using the commercial polymeric coating Mix&Go™. Sandwich type immunoassays with amperometric signal amplification through the use of poly-HRP-streptavidin conjugates and H O as HRP substrate and hydroquinone as redox mediator were implemented. Upon optimization of the experimental variables affecting the immunosensor performance, the dual immunosensor allows ranges of linearity extending between 0.5 and 100 pg/mL and from 1 to 200 pg/mL for IL-1β and TNF-α, respectively, these ranges being adequate for the determination of the cytokines in clinical samples. The achieved limits of detection were 0.38 pg/mL (IL-1β) and 0.85 pg/mL (TNF-α). In addition, the dual immunosensor exhibits excellent reproducibility of the measurements, storage stability of the anti-IL-Phe-DWCNTs/SPCE and anti-TNF-Phe-DWCNTs/SPCE conjugates, and selectivity as well as negligible cross-talking. The dual immunosensor was applied to the simultaneous determination of IL-1β and TNF-α in human serum spiked at clinically relevant concentration levels and in real saliva samples.
We develop detachable “Cavitas sensors” to apply to the human oral cavity for non-invasive monitoring of saliva glucose. A salivary biosensor incorporating Pt and Ag/AgCl electrodes on a mouthguard support with an enzyme membrane is developed and tested. Electrodes are formed on the polyethylene terephthalate glycol (PETG) surface of the mouthguard. The Pt working electrode is coated with a glucose oxidase (GOD) membrane. The biosensor seamlessly is integrated with a glucose sensor and a wireless measurement system. When investigating in-vitro performance, the biosensor exhibits a robust relationship between output current and glucose concentration. In artificial saliva composed of salts and proteins, the glucose sensor is capable of highly sensitive detection over a range of 5–1000 µmol/L of glucose, which encompasses the range of glucose concentrations found in human saliva. We demonstrate the ability of the sensor and wireless communication module to monitor saliva glucose in a phantom jaw imitating the structure of the human oral cavity. Stable and long-term real-time monitoring (exceeding 5 h) with the telemetry system is achieved. The mouthguard biosensor will be useful as a novel method for real-time non-invasive saliva glucose monitoring for better management of dental patients.
Background: Exosomes are 30-100 nm membrane vesicles of endocytic origin produced by numerous cells. They can mediate diverse biological functions, including antigen presentation. Exosomes have recently been shown to contain functional RNA, which can be delivered to other cells. Exosomes may thus mediate biological functions either by surface-to-surface interactions with cells, or by the delivery of functional RNA to cells. Our aim was therefore to determine the presence of RNA in exosomes from human saliva, plasma and breast milk and whether these exosomes can be taken up by macrophages. Method: Exosomes were purified from human saliva, plasma and breast milk using ultracentrifugation and filtration steps. Exosomes were detected by electron microscopy and examined by flow cytometry. Flow cytometry was performed by capturing the exosomes on anti-MHC class II coated beads, and further stain with anti-CD9, anti-CD63 or anti-CD81. Breast milk exosomes were further analysed for the presence of Hsc70, CD81 and calnexin by Western blot. Total RNA was detected with a Bioanalyzer and mRNA was identified by the synthesis of cDNA using an oligo (dT) primer and analysed with a Bioanalyzer. The uptake of PKH67-labelled saliva and breast milk exosomes by macrophages was examined by measuring fluorescence using flow cytometry and fluorescence microscopy. Results: RNA was detected in exosomes from all three body fluids. A portion of the detected RNA in plasma exosomes was characterised as mRNA. Our result extends the characterisation of exosomes in healthy humans and confirms the presence of RNA in human saliva and plasma exosomes and reports for the first time the presence of RNA in breast milk exosomes. Our results also show that the saliva and breast milk exosomes can be taken up by human macrophages. Conclusions: Exosomes in saliva, plasma and breast milk all contain RNA, confirming previous findings that exosomes from several sources contain RNA. Furthermore, exosomes are readily taken up by macrophages, supporting the notion that exosomal RNA can be shuttled between cells.
Oral Diseases (2011) 17, 345–354 For the past two decades, salivary diagnostic approaches have been developed to monitor oral diseases such as periodontal diseases and to assess caries risk. Recently, the combination of emerging biotechnologies and salivary diagnostics has extended the range of saliva‐based diagnostics from the oral cavity to the whole physiologic system as most compounds found in blood are also present in saliva. Accordingly, saliva can reflect the physiologic state of the body, including emotional, endocrinal, nutritional and metabolic variations and acts as a source for the monitoring of oral and also systemic health. This review presents an update on the status of saliva diagnostics and delves into their applications to the discovery of biomarkers for cancer detection and therapeutic applications. Translating scientific findings of nucleic acids, proteins and metabolites in body fluids to clinical applications is a cumbersome and challenging journey. Our research group is pursuing the biology of salivary analytes and the development of technologies for detection of distinct biomarkers with high sensitivity and specificity. The avenue of saliva diagnostics incorporating transcriptomic, proteomic and metabolomic findings will enable us to connect salivary molecular analytes to monitor therapies, therapeutic outcomes, and finally disease progression in cancer.