The challenge for successful use of saliva for medical diagnostics resides in maximizing the advantages and overcoming the disadvantages of using saliva/oral fluids. Compared to serum samples, the volume of saliva that can be obtained is relatively limited and disease-specific salivary biomarkers are still largely unknown. However, saliva can be obtained by patients themselves or by personnel with little medical training. Furthermore, saliva collection is associated with less stress and discomfort to the patient/donor. Therefore, saliva-based diagnostics can be applied in medically disadvantaged areas or non-conventional medical settings, such as in developing or under-developed countries, remote rural areas, patients’ homes, as well as in the dentist’s office or neighborhood pharmacy.
Recent studies have demonstrated improvement of sensitivity and specificity using a combination of multiple biomarkers instead of a single biomarker in disease detection 12;13
. Therefore, a successful saliva/oral based diagnostic should provide accurate, non-invasive, disease-specific, multi-analyte and rapid outcome measurements, as well as be portable and cost-effective. Current efforts emphasize the discovery and validation of disease biomarkers in saliva, the development of multiplexed nanotechnologies (lab-on-a-chip) for point-of-care, and their ultimate translation into the real world through an industrial partner ().
Development of analytical technologies in the post genomic era has allowed for large scale identification of proteins/peptides (proteome) and ribonucleic acids (RNA; transcriptome), and their functions/structures in cells and fluids. The high throughput proteomic studies have catalogued at least 1166 proteins in the major salivary gland secretions, of which 914 are recovered from parotid and 917 from submandibular/sublingual ductal saliva, with 57% of these proteins present in both glandular saliva 14
. The proteome of human minor salivary gland secretion showed 56 proteins, 12 of these proteins have never been indentified in the glandular saliva 15
. Analysis of human whole saliva and plasma has identified a total of 1939 proteins in whole saliva, with 740 proteins in glandular saliva proteomes and 597 saliva proteins in plasma 16
. More surprisingly, the salivary transcriptome (RNAs) has been discovered using microarray profiling in recent years. It is estimated that approximately 3000 messenger RNAs (mRNAs) have been identified in cell-free whole saliva. Most recently, the presence of microRNA (miRNA; ~50) was also discovered in whole saliva. Unlike mRNA, miRNA consists of 18–24 nucleotides transcribed from non-protein coding genes and regulates protein translation through an RNA-induced silencing complex (RIST) 17
. These advances have provided a large number of salivary molecular targets, e.g., proteins and RNAs, for disease biomarker discovery. Several investigators have already attempted to use high throughput technologies and current salivary proteomic and transcriptomic knowledge for biomarker discovery in the areas of oral cancer 17;18
, breast cancer 19
, periodontal diseases 20;21
, cardiovascular disease 13
and Sjögren’s syndrome 22;23
In the past few years, multiplex biomarker detection systems have emerged through remarkable progress in the development of lab-on-a-chip (LOC) and point-of-care (POC) technologies 24
. The goal of these efforts is to build automated, miniaturized, and multiplexed platforms for rapid assays and readout. In general, the principles of conventional enzyme-liked immunosorbent assay (ELISA) and/or nucleic acid hybridization are applied often with either electrochemical sensors 12
or a microbead reactor 13;25
. The electrochemical approach uses gold electrode arrays (multiplex chips) in which one set of electrodes (i.e., working, counter and reference electrodes) is used for one analyte measurement applied with the cyclic square wave electrical field to facilitate chemical reaction, followed by amperometric readout 12
. The UCLA School of Dentistry “UCLA Collaborative Oral Fluid Diagnostic Research Center” is the leading institute for this nano/micro-electrical-mechanical development. Alternatively, the microbead reactor system developed by the Texas-Kentucky Saliva Diagnostic Consortium consists of porous bead sensors consisting of a nano-net of agarose fibers serving as a chemical reaction matrix sequestering and concentrating analytes. The beads are placed in a microchip holder with each bead serving as a 3-dimensional reactor. Multiple beads can be placed in the holder with modulation of their analyte specificity through the capturing antibody they are conjugated to, providing a multi-analyte testing platform. The reaction reagents are delivered through a self-contained microfluidic infrastructure and the measurement is reported by nano-particle fluorescent particles or dyes that are conjugated to detecting antibodies. This approach results in increased signal to noise ratios and amplification several orders above conventional assays 24
The Texas/Kentucky Saliva Diagnostics Consortium is in the forefront of developing 3-D bead saliva/oral fluid diagnostics for cardiovascular, cancer, and periodontal diseases 13;26;27
. As compared to other systems, this approach is cost-effective and more flexible than any other LOC system reported in the literature. For example, the bead reactor can be replaced with a thin-polymeric membrane for analyzing cell isolation/trapping from saliva or serum or oral brush biopsy samples, e.g., oral cancer cell studies.
Current efforts to develop a saliva-based nano-biochip test for acute myocardial infarction (AMI) at the-point-of-care, particularly in the emergency settings, and for cytological diagnosis of oral cancers are briefly described below.