Salivary Sensors in Point-of-Care Testing

The key technologies for realizing diagnosis of systemic diseases using saliva samples are biochips, biosensors, and biomarkers. Chemical salivary test (salivary using tremendously point-of-care testing


Introduction
Saliva has attracted attention as a human sample that can be collected noninvasively not only for the diagnosis of oral diseases (1) but also for the potential diagnosis of systemic diseases. (2)(3)(4) Considerable time has passed since it was fi rst predicted that saliva could become a substitute for blood as a medical test sample (sample). The time may have fi nally arrived for this prediction to be realized, that is, when saliva can be used in point-of-care testing (POCT). Examples of POCT have become familiar in recent years. For example, the self-monitoring blood glucose meter is now widely used by diabetics and disposable pregnancy testing kits using urine samples have become well established as over-the-counter (OTC) products sold in drug stores. Thus, clinical tests that were previously limited to specialist testing centers could now be replaced by POCT using samples other than blood.
In developing POCT devices, it is necessary to consider eliminating mental and physical pains associated with sampling in addition to the risk of infection from the samples. In the roadmaps for drug development and diagnosis that are being drawn up in advanced countries, technologies with semi-and non-invasive approaches to sample collection and processing could be realized around 2015.
The elemental technologies that are required to realize such a diagnostic methodology for systemic diseases using saliva samples are as follows: (i) Biochip: used for collecting and concentrating saliva samples through a simple and hygienic process to obtain the volume necessary for testing; (ii) Biosensor: used as a rapid, low-cost, high-sensitivity analytical technology for biomarkers; (iii) Biomarker: a chemical or substance that can be effectively used for the prevention and diagnosis of diseases, since they are strongly correlated to the severity of the disease. Technical consideration of the infl uences of ingredients, inhibitors or competitive chemicals is required. One possibility for the realization of such a diagnostic methodology is based on the development and widespread use of micro-electromechanical systems (MEMS) developed in the semiconductor industry together with the extensive development of bioanalytical techniques employing high-specificity and high-selectivity molecular recognition mechanisms of enzymes, antigens/antibodies, mRNA and DNA. (5) In other words, owing to collaborations between medical and engineering researchers, above criteria (i) and (ii) are no longer constraints.
This review focuses on the current status and future prospects of diagnostic methodologies using saliva samples for detection of systemic diseases. The direction of new applications for salivary sensors is discussed by introducing commercially available salivary tests for oral and systemic diseases. Table 1 shows the advantages of saliva sampling in POCT for systemic diseases. The principal advantage of salivary test over chemical blood test is its safety. The use of needles in blood collection using cannot be completely free of danger from infection by disease-causing viruses. Since salivary test is a noninvasive technique, then patients who are currently restricted from using needles, such as children, the aged, and hemophiliac patients, will be afforded a number of advantages in self-monitoring. Next, unlike blood, saliva can be self-collected, and unlike urine, saliva can be collected any time. These are the important conditions that POCT must meet in order to make on-site testing possible at home and in the workplace. Furthermore, it is particularly useful for mass screening tests. The infl uences of ingredients, viscosity and color intensity of each sample on analysis accuracy are different from those of biomarkers. Table 2 shows approaches to the diagnosis of systemic diseases and body conditions using salivary biomarkers. (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24) Some salivary biomarkers that could possibly be used for diagnostic methodologies have already been studied and even partly applied to clinical use. They can be classifi ed in terms of origin into antigens/antibodies, hormones, neurotransmitters, genes, enzymes and xenobiotics. Among those listed above, only antibody testing for HIVs (HIV-1 and HIV-2) has been made available commercially (OraSure Technologies, Inc., PA). Development of methods for the proteome (entire complement of proteins expressed by a genome) and transcriptome (set of all messenger RNA (mRNA) molecules) analyses of saliva is currently progressing. Saliva mRNA Table 2 Approaches to the diagnosis of systemic diseases and body conditions using salivary biomarkers.

Biomarker
Disease and condition Reference Classifi cation Chemical Antigen and antibody HIV AIDS Ishikawa et al. (6) Hepatitis C Hepatitis Champion et al. (7) Hormone Cortisol Stress-induced diseases Vining et al. Olff et al. (11)

Phenytoin
Drug abuse Fu et al. (23) Cocaine, Codeine Prohibited drug, Narcotic drug Kacinko et al. (24) HIV, human immunodeficiency virus; AIDS, acquired immune deficiency; DHEA, dehydroepiandrosterone; E2, estradiol; Pg, progesterone; mRNA, messenger ribnucleic acid; DNA, deoxyribonucleic acid profi les have been obtained using a DNA chip (HG U133A array, Affymetrix, Inc., CA) containing 22,215 human gene cDNA probe sets. (15) Between 2,521 and 3,363 saliva mRNAs have been reliably detected from participants. For many stress-induced diseases, no objective or quantitative diagnostic standards have yet been established. Some biomarkers that are directly or indirectly related to the sympathetic nervous system and endocrine system can signifi cantly change in concentration depending on the degree of stress and thus be labeled as stress markers. Therefore, stress markers such as cortisol have been studied as a diagnostic index for post-traumatic stress disorder (PTSD), chronic fatigue syndrome (CFS) and irritable bowel syndrome (IBS). In the United States, a business model for the stress testing of commercial pilots is ready for launching. In this business model, the kit is fi rst purchased through the internet. The user then mails in a saliva sample that he/she collected himself/herself and the test results are returned showing the number of stress markers and whether or not the absolute values are within standard range.
Xenobiotics have typically been used for physical condition evaluation rather than for the diagnosis of diseases. Therapeutic drug monitoring (TDM) for antiepileptic drugs and investigations into smoking and drug abuse are typical uses of such applications. A drug-abuse testing system (RapiScan, Cozart Bioscience Ltd., UK) has been used as a method of on-site testing by Immigration Offi cers at airports in Europe. Table 3 and Fig. 1 respectively show the principles and schematic diagram of the sensor devices used for the detection of salivary biomarkers. The specifi city of these procedures increases in the following order, enzymatic method, antigen-antibody method, hybridization method; however, the cost of the tests increases in the same order. Studies using salivary biomarkers have become very popular following sales of a series of enzyme-linked immunosorbent assay (ELISA) kits for use with saliva samples by Salimetrics, LLC (PA, USA) and other companies. The gold colloid method ( Fig.  1(a)) makes a low-cost qualitative test possible by exhibiting the color reactions of ELISA on a visible line on a biochip. Enzyme sensors ( Fig. 1(b)), immunosensors and surface plasmon resonance (SPR, Fig. 1(c)) (25) exhibit the color reactions of the ELISA process by way of electro-chemical or optical phenomena, and thus, high-sensitivity and continuous measurements are possible. A nanoscale optical biosensor based on localized surface plasmon resonance (LSPR) spectroscopy has been developed to monitor the interaction between antigens and antibodies. (26) DNA chips (DNA microarrays) are a new tool used to identify mutations in genes. A DNA chip, which consists of a small glass plate encased in plastic, is manufactured similarly to a computer microchip ( Fig.  1(d)). The surface of each chip contains thousands of short, synthetic, single-stranded DNA sequences, which together add up to the normal gene in question when competitive hybridization occurs between the immobilized synthetic cDNA/cRNA strands and mRNA/DNA in a sample. Because chip technology is still relatively new, it is currently only a research tool. In order to consider new approaches to diagnostic methodologies using salivary biomarkers, some novel devices that are already on the market are introduced.

Salivary Test of Oral Diseases
A novel POCT platform (DentoAnalyzer, Dentognostics GmbH, Germany) to quantify micro-organisms that cause dental infections and inflammatory markers refl ecting oral disease status has been put on the market in Germany (Fig. 2). (27) This system is based on sandwich immunoassay technology, using a disposable cartridge and is used for the quantitative analysis of salivary markers as measured for the absorbance of light. Thus, the principle is almost the same as that of ELISA. The main component is a small unit that is easily available in dental clinics. Polyhorseradish peroxidaseconjugated antibodies have been developed for detection of dental infections and infl ammatory markers, which results in high sensitivity.
The above assay enables a user to quantify 500 colony-forming units of Streptococcus sobrinus (S. sobrinus) per ml of saliva as a dental infection marker. Streptococcus mutans (S. mutans) (28) is considered to be the major cause of dental caries. S. mutans and S. sobrinus have been widely detected in all ethnic groups. On the other hand, matrix metalloproteinase-8 (MMP-8) is analyzed as a dental infl ammatory marker secreted by periodontium cells. MMP-8 has been identifi ed as a major tissue-destructive enzyme of the extracellular matrix in periodontal diseases.
The diagnosis of oral diseases using MMP-8 has been focused on by a relatively large number of researchers. A micro-total analysis system (µTAS) that utilizes MEMS technology to create micro-fl ow channels and reaction vessels on a biochip to analyze biomarkers from minute volumes of test samples has been fabricated. A prototype µTAS for MMP-8 analysis (Sandia Corporation, NM) enables the user to specifi cally detect Fig. 2. Immunoassay-based diagnostic technology to quantify micro-organisms causing dental infections and infl ammation (DentoAnalyzer, Dentognostics GmbH, Germany).
MMP-8 with a fl uorescence-labeled anti-MMP-8 antibody, and then to separate it by electrophoresis for further fl uorescence analysis. This device enables the quantifi cation of MMP-8 within only 10 min from only 20 µl of saliva sample. (29) This method exhibits a favorable linearity of R 2 = 0.979 compared with conventional ELISA.

Diagnosis of virus infections
The DNA and RNA detection of viruses by reverse transcription-polymerase chain reaction (RT-PCR) has already been used for the diagnosis of infections (genetic diagnosis). However, owing to the long time required for the preparation and amplifi cation of DNA/RNA, rapid diagnosis is impossible. Therefore, in an attempt to realize its practical use in POCT, efforts have been made to considerably shorten the time necessary for analysis using immunoassays.
As a diagnostic methodology for systemic diseases, a prototype rapid antigen test for the on-site detection of respiratory syncytial virus (RSV) infection has been developed by OraSure Technologies Inc. (UPlink, PA, Fig. 3) (30) RSV belongs to Paramyxoviridae, the same family as the parainfl uenza, measles, and mumps viruses. RSV has been reported worldwide and causes an infection that affects the lungs and causes infant bronchiolitis and pneumonia.
The currently available UPlink was originally developed for the on-site testing of oral samples for drug abuse. UPlink consists of two kinds of disposable devices (UPlink collector and assay cassette) and a monitor with a near-infrared laser (UPlink analyzer). UPlink is based on sandwich immunoassay technology using an RSV monoclonal antibody. Qualitative tests are being conducted to determine whether a test line of a monoclonal antibody can be seen on a cellulose pad placed on the assay cassette. In this way, the need to develop a rapid diagnosis technology for infection arises from the necessity of preventing infectious diseases spreading in today's borderless era. Fig. 3. Prototype of rapid antigen test for the detection of respiratory syncytial virus infection (UPlink, OraSure Technologies, Inc., PA, Ann. N. Y. Acad. Sci. 1098 (28) ).

Human stress evaluation
It is widely accepted that psychological stress can produce physiological effects that are similar to those produced by physical challenges. Cortisol is an established biomarker of the hypothalamic-pituitary-adrenocortical (HPA) axis. Salivary cortisol concentration closely correlates to serum cortisol concentration. (31) In contrast, such a well-characterized salivary biomarker related to the activity of the sympathetic nervousadrenalmedullary (SAM) system is still missing. Salivary α-amylase activity (SAA) has been found to increase slightly with salivary fl ow rate, and large increments in amylase concentration have been observed during sympathetic control by Speirs et al. (20) Medication with betablockers signifi cantly suppresses the secretion of SAA, which provides direct evidence of the sensitivity of SAA to changes in adrenergic activation. (32) Currently, it is considered that measurement of SAA is useful for evaluating the SAM system.
To realize a hand-held monitor of the sympathetic nervous system, a completely automated analytical system for measuring SAA using a dry-chemistry system was developed (Amylase monitor, Nipro Co., Japan, Fig. 4). (33) This monitor was made possible by the fabrication of a disposable test strip equipped with built-in collecting and reagent papers and an automatic saliva transfer device. Within an SAA range of 10 -150 kU/l, the calibration curve for the hand-held monitor showed a coeffi cient with R 2 = 0.97. Accordingly, it was demonstrated that the hand-held monitor enables a user to automatically measure SAA with high accuracy with only 30 μl of saliva within a minute from collection to measurement completion.
It is reported that SAA increases more signifi cantly than the level of the major chronic-stress marker cortisol, suggesting that it is a better index of acute stress. (34) Fig. 4. Hand-held monitor for sympathetic nervous activity using salivary amylase activity (salivary amylase monitor, Nipro Co., Japan).

Conclusion
Salivary tests have overwhelming advantages over blood tests in terms of safety and the ability for self-collection of samples. However, if we only consider these advantages alone, it is not easy to establish a diagnostic methodology for systemic diseases. It is considered that the key to the realization of a practical salivary testing is achieving greatly enhanced diagnostic accuracy for this kind of test compared with conventional technologies, or providing useful new information for medical treatment. In other words, the key is the new usage of biomarkers. The development of a rapid, low-cost and highsensitivity analytical technology for saliva samples has progressed signifi cantly. This could promote the application of saliva testing to the rapid diagnosis of stress-induced diseases and viral infections, for which we have as yet no standard diagnosis based on an objective and quantitative index. The advent of POCT could provide a way for reaching this goal.