We are proposing to make a rapid device for detecting cortisol levels in interstitial fluid (ISF). A rapid cortisol test developed during our NIH funded Phase I (1R43MH085474-01, SBIR) proposal in collaboration with the University of South Florida (USF). [1
,Arya et. al. 2010, 2
Arya et. al, 2010] will be integrated in this device to develop a portable test for real-time and continuous measurements of personal exposure to psychosocial stress via measuring cortisol levels in ISF. Sampling of biochemical markers in the body via blood or saliva plays an integral role in the diagnosis and management of several diseases tied to cortisol levels in the body. However, due to its invasive nature blood sampling is less suitable for use in nonclinical settings especially when a continuous measurement is desirable. Saliva, on the other hand, requires constant compliance from the patient. ISF is especially attractive for cortisol monitoring, as it can be drawn continuously from the dermis through an ablated stratum corneum by simply applying a small amount of vacuum. ISF offers less invasive and compliance free method for monitoring levels of cortisol in the body. ISF is an extra cellular fluid that surrounds the cells in the human body and consists of small and moderate sized molecules, including glucose, ethanol, and cortisol. In composition, ISF is similar to blood plasma. The homeostatic feedback loop in the body ensures that these molecules have a direct correlation to the concentration of molecules in blood [3
Stout et. al., 2004; 4
Bentle and Thomas, 1997]. Metabolites and proteins move into ISF as they move from capillaries to cells. Consequently, the metabolite concentration in ISF is correlated to their concentration in the capillaries. The difference in concentration is based in part on molecular weight [5
Fischer, 1994 and 6
Sternberg, 1995]. ISF can be harvested using a minimally invasive method. The site of ISF extraction can be almost anywhere on the human body (arms, abdomen, legs) with no loss of accuracy. The application of vacuum pressure to extract ISF significantly reduces the integration time required for accurately determining the metabolite concentration. The lag between blood and ISF levels can contribute to measurement error in continuous monitoring systems. 3
Stout et. al., showed that the modulated pressure application mitigated the ISF physiological error by an average of 95%. Clinical tests involving diabetic patients have shown that the correlation between ISF glucose concentration and blood glucose levels is as high as 0.90 in the 60–400 mg/dl glucose range [7
Gebhart, et. al., 2003]. Using this methodology, Gebhart et al.
, reported correlations of 0.87 and 0.95 between blood and ISF glucose. This technique has also been successfully used for continuous monitoring of glucose [8
Daniloff, 2005]. In general, small to moderate sized molecules, including glucose and ethanol, are found in ISF in the same proportion as in blood. Thus, periodic calibration using blood sampling is not required to obtain the concentration of these metabolites from ISF (larger molecules such as certain lipids also are detectable in this body fluid, but at a reduced concentration relative to blood [9
Bentle and Thomas, 1997 and 10
Cortisol has been identified as a key element in the psychobiology of the stress response and therefore it can be used as a biomarker of stress. Cortisol has a range of roles in the body. It helps break proteins, glucose, and lipids, maintain blood pressure, and regulate the immune system. Inadequate amounts of cortisol can cause nonspecific symptoms such as weight loss, muscle weakness, fatigue, low blood pressure, and abdominal pain. Too much cortisol can cause increased blood pressure, high blood sugar, obesity, fragile skin, purple streaks on the abdomen, muscle weakness and osteoporosis. Abnormal levels of cortisol may also influence certain pathological conditions such as type 2 diabetes [11
Abdallah et al.
, 2005; 12
Wren and Garner, 2005], constant stress, obesity, and metabolic syndrome. In order to understand any of these factors doctors often have to order a cortisol test. Typically blood will be drawn from a vein in the arm (blood drawn at 8 am, when cortisol is at its peak and then at 4 pm, when level should have dropped), but sometimes urine (24-hour urine test, usually requires collecting all the urine produced during a day and night) or saliva (patients compliance is required to ensure they have not had anything to eat or drink 20 minutes before sample collection) may be used to perform a test. All of these methods of sample collection and testing are not only inconvenient, protracted, and painstaking but also take time to get the results back from the doctor or a laboratory.
Current methods for cortisol testing include the saliva test [13
Petkus et al.
, 2006], the Fluorometric assay [14
Appel et al.
, 2005], Fluorescence Polarization [15
Cullum, et. al., 2006] and Reverse Phase Chromatography [16
,Gatti et al.
, 2005]. These methods are, however, limited in sensitivity, time of analysis and cost [17
,Cook 1997; 18
Kaptein et al.
, 1997]. None of these methods are rapid and consequently results for cortisol cannot be obtained sequentially. Salivary Cortisol concentrations have become a valuable tool for both basic scientists and clinicians [19
Papanicolaou et. al, 2002]. Testing of salivary cortisol has become the method of choice despite many disadvantages. However, none of these methods will allow us to get the nighttime minimum value which can identify the precise point in sleep cycle at which cortisol begins its morning cycle. To date, no rapid, reliable real-time and sequential test is available for cortisol. The major issues limiting the usefulness of prevailing tests are compliance, collection device and contamination of saliva sample. A single time assessment of cortisol level is not ideal as cortisol fluctuates throughout the day due to circadian rhythms and therefore results will differ in “early birds” and “night owls”. Researchers take samples at a specific time during the day, but patients are often non-compliant which can invalidate their results and mask potential differences between subject groups.
In this work, we demonstrate a new technique for ISF sampling for the purpose of near to continuous monitoring of cortisol concentration in the human body over 24 hours. No previous correlation exists for this molecule between ISF and blood or saliva. Therefore, our current work will help to fill up this gap. Our proposed device will be a portable diagnostic tool and will be capable of non-invasively measuring cortisol in real time in ISF. Cortisol sensor being described here will automatically provide sequential cortisol data from the patient over 24 hours with minimal compliance from the patient. Further, a pilot clinical study has been designed to validate the feasibility of making in-vitro measurement of cortisol using the sensor system being developed in collaboration with USF. We developed this electrochemical Impedance system (EIS) for sensing cortisol levels in ISF and saliva [1, 2
Arya et. al., 2010]. Our ultimate goal is to combine the ISF harvesting design with the cortisol sensor system in order to develop a rapid sensor that can sequentially measure cortisol in real-time.
A less invasive and bloodless method for metabolite monitoring involves sampling ISF, which is an extra cellular fluid that surrounds the cells in the human body. ISF is present just below the skin, but the low permeability of the epidermal keratinized layer (the stratum corneum or SC) blocks the permeation of the fluid through the skin. In this paper, we briefly describe a minimally invasive technique to access the ISF through the skin and measure levels of cortisol in the collected fluid. We have used a minimally invasive method for continuously extracting ISF from SC [U.S. patent 6,183,434; Altea MicroPor(TM) Laser]. This method entails focusing a near infrared laser (low-energy) on a layer of black dye material affixed to the skin by an adhesive (-Right, 20
Venugopal et al.
, 2008). The interaction of the light at the dye layer causes a pyrotechnic event; thereby creating tiny pores (called micropores) in SC (-Left and Middle). The diameter of the micropores is approximately equal to that of a human hair. The micropores only penetrate the SC and, hence, this procedure is essentially painless. ISF is drawn through these micropores continuously by application of a small amount of vacuum pressure.
Figure 1 Left: Cross-section of a micropore. Note that the pore does not extend to the dermal layer. Middle: Four pores relative to the size of a penny. Right. Micropores are made using a handheld laser source that is focused on black dye attached to the skin. (more ...)