The Correlation of Oxygen Level to the Optical Response of Porphyrin This is a mini-treatise on the utility of Porphyrin as a high accuracy, small Oxygen sensor. When this compound is placed at the end of a fiber optic lead, this creates a sensor interface that is smaller than any conventional Clark Cell in the laboratory marketplace. The Matrix, the Porphyrin in a binder, applied to the end of a 1 mm fiber optic has been used to measure the Oxygen content of an assay vessel 100 micro liters in volume. Introduction Porphyrin is a compound that is related to plant and animal energy transfer mechanisms. In plants it is associated with photosynthesis, in animals it has connections with the transfer of Oxygen in blood. It has been studied since the 1940's in regard to its property of fluorescence when excited by an ultra-violet source. During these studies, it was found that Porphyrin exhibits a correlation between its fluorescence and the level of Oxygen saturation found in its environment. Fluorescence is the optical property of a compound by which it emits light by virtue of having been first stimulated by light. The common experience of this phenomenon is found in the Black Light exhibits of minerals in natural history museums. The Black Light, an ultra-violet source, stimulates the rock samples usually displayed inside a darkened or semi-darkened room. The blueness of the light is such that it is a very poor general illumination for common viewing tasks. Thus, the perception of viewing this light indirectly is that it is not very bright. However, when this light strikes the sensitive minerals, they display vivid colors in response. Another common experience of this phenomenon is found in the Day-Glo posters quite prevalent some years ago. In this application, it is the paint of certain colors that is fluorescent. For Porphyrins, this phenomenon is called a red-shifted response. That is, when an ultra-violet source excites Porphyrin, it absorbs that energy and then releases it as a red light emission. Thus, the source and the response are separated in wavelength, or color, and this allows for an apparatus to distinguish between the source of excitation and the consequent response of that excitation. It is in Porphyrin's response, and that correlation to Oxygen saturation of its environment, that makes for a new sensor source. This correlation is notable as the "quenching" effect Oxygen has on Porphyrin's ability to emit a red-shifted response. That is, Oxygen has the effect of turning off, or at least radically reducing, the red-shifted emission. The Oxygen Sensing System The correlation of Oxygen to this novel sensor requires a closer scrutiny of this red-shifted emission. The excitation source is a pulse of ultra-violet light. It is funneled into a 1 mm fiber optic that is terminated in a target composed of Porphyrin in a matrix suitable for sensor application. This target is illuminated by the pulse for roughly 1 microsecond. The target is also viewed by a second fiber optic which applies the Porphyrin's response emission back to an optical detector. By design, the excitation and response of Porphyrin are separated in path, color and time. As of yet undisclosed is this separation in time. Porphyrin being excited by a pulse of ultra-violet light does not immediately convert this to the red-shifted emission. As long as the light persists, the Porphyrin remains excited and any emission is uncorrelated to the Oxygen in the environment. Upon the cessation of excitation, the Porphyrin relaxes and releases this energy in a red pulse. The correlation of Oxygen levels is found in the time based characteristics of this red-shifted emission pulse. Tau, the indirect measure of Oxygen correlated to pulse time decay The red-shifted emission pulse is a quick peak with a rapid decay. The time for the red light emitted from the Porphyrin target to decay to undetectable levels varies from 10's of microseconds to 100's of microseconds depending upon the level of Oxygen content in the environment of the target. The illustration that follows displays the truncated pulse of red-shifted response to the ultra-violet excitation. The response pulse's peak is of no importance as it is the slope of the decay curve that is correlated to Oxygen As may be noted above, the red-shifted emission persists for a considerable time (in microsecond time scales). However, there is only a short interval over which precise measures may be made which are rarely beyond the 200 microsecond mark. Tau is the slope of these curves expressed as the time taken by the sensor's red-shifted light to decay from one brightness level to a lower one. The curves above can be described as exhibiting Tau's of 15 to 60 microseconds depending upon the Oxygen levels in the environment of the sensor. That is, for a highly Oxygenated sensor, the light decays from a bright level to a level 1/3rd as bright within 15 microseconds. On the other hand, for a relatively low Oxygenated sensor, the light takes much longer, 50 to 80 microseconds, to decay the same amount. The Oxygen System Calibration The correlation of Oxygen saturation to Tau was performed with a highly accurate Clark Cell in a tracking configuration. Both sensors were presented the same conditions by their introduction to a closed loop system consisting of a computer based gas mixer feeding a circulating temperature controlled water bath. The correlation is illustrated as follows. As may be noted, the correlation is not linear, but it is strong. The spread of data is more due to the inherent noise of the Clark Cell and its slow time response rather than due to the Porphyrin target and its associated circuitry. All tests suggest that although the Clark Cell must be acknowledge as being the primary standard; that the Porphyrin sensor is superior in every respect. Applications of the Porphyrin as an Oxygen Sensor The small design of the Porphyrin matrix as a target on a 1 mm diameter Fiber Optic lead allowed for the creation of very fast biological assays. A simple example is in the creation of a cell culture inside a sealed environment, monitored by a Fiber Optic based Porphyrin Oxygen sensor. The cell culture population grows over time and through that growth consumes the available Oxygen. The Oxygen content of the sealed environment is exhausted over time and this is clearly exhibited in the Tau data for this sensor: The data above presents evidence of vigorous growth followed by a knee in the curve and then a complete cessation. these last two artifacts can be seen as the population having consumed the last of the available Oxygen before dying. The complete process took roughly 2 hours for the size of population started. With this confirmation of observing cellular activity (indirectly as a function of respiration), the next step was to observe how the sensor responded to variations in initial population density. By employing the same configuration, the population was varied by three orders of magnitude through serial dilution of an initial stock of cell culture. The results are interleaved in the graph below. The four curves each represent a population 1/10th of the curve to their left. Thus, an analysis may be performed on the order of less than one hour on the basis of observing the complete curve to the extinction of the population. The following data is an enlarged view of that above. It portrays the clear, and divergent paths taken by two dilution's in the early stages. From this data, it is evident that qualitative analysis may be performed in as little as 10 minutes. That the Porphyrin sensor is able to resolve large dynamic changes is established above. However there is also the question about how well does it resolve small differences. The following data presents two cultures whose initial populations vary by only 2% between them. The two responses (the consumption of Oxygen by the cell cultures) are clearly divergent by 20 minutes as seen above. With a closer look at the early data below, it could be argued that a qualitative result may be found within 10 minutes. Porphyrin Oxygen Sensor System Specifications These specifications are as observed, not projected. Sensor size: 1 mm square/diameter; Environment: Dissolved or Free Gas, up to 50 Degrees Celsius; Oxygen saturation range: 0 - 100%; Time for sensor to settle to within 10%: less than 1 second; Time for sensor to settle to within 1%: less than 10 seconds; Time for sensor to settle to within 0.1%: less than 1 minute; Accuracy: 0.05% or better; Spurious responses: no offsets, no drift, no hysteresis; Ruggedness: far superior to glass electrodes. With the recent emergence of powerful, Blue LEDs, there is the potential for developing a very small, modestly accurate Oxygen survey instrument. These LEDs offer an alternative in excitation sources as well as reducing system packaging constraints considerably. Conclusion The Porphyrin sensor's size is one of its key assets. It has been employed in bio assays with small samples sizes of 100 microliters and is easily applicable to standard micro well plates of automated assay equipment. The method of affixing the Porphyrin target to a fiber optic is not required, as the target may be constructed and added to an assay separately if there are suitable optical paths to allow the target to be illuminated by the source and viewed by the detector. As most well plate designs are clear plastic, attention to placement of such a target allows for alternatives to fiber optic illumination and detection. As may be anticipated, larger targets can result in faster assays to high accuracy. As for practical applications: BOD measurements can be performed in hours instead of days. Toxicology testing can be performed through the indirect measure of cellular Oxygen consumption. The use for Blood-Gas chemistry is also an obvious market. Other areas of study included the detection of caries on tooth enamel as part of a general dental health measurement system. Porphyrin presents a stable, accurate, and reproducible sensor that can perform in every Oxygen sensing application found using a Clark Cell, and many Oxygen sensing applications that are entirely hostile to this old technology.