Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1998-05-08
2001-07-17
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S161000, C436S529000, C436S530000, C436S531000, C436S534000, C436S536000, C435S287300, C435S291400, C435S288600, C422S051000, C422S069000, C422S070000, C210S198200, C210S656000, C210S659000, C250S458100, C250S459100, C250S461100, C250S462100
Reexamination Certificate
active
06261848
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates in general to sensors and, more particularly, to a hand held, self-contained, automatic, low power and rapid sensor platform for detecting a plurality of analytes.
While the invention can be used to detect a wide variety of analytes, the initial embodiment was developed for use against aflatoxin which is formed from a fungus commonly found in many grain products and peanuts. Studies have proven that ingesting aflatoxin can cause serious health problems. The FDA is aware of this health risk and has set tolerance levels for aflatoxin for United States products such as flour and milk.
However, high levels of aflatoxin do still exist in food in remote areas and foreign countries, where it is also used in weaponry. It is these unacceptable levels in food and the potential use in weapons that have prompted investigations into a portable sensor for aflatoxin and, by extension, to other analytes of interest as well.
There currently exist two different measurement techniques for aflatoxin levels. The easiest and most portable method currently available uses a “wet chemistry” approach. This method uses a paper that changes color when immersed in a minimum concentration of aflatoxin. Currently available test papers are for minimum levels of 5 parts per billion (ppb) and 20 ppb.
The second, less portable method of measuring aflatoxin levels uses affinity chromatography and a series of manual fluid wash and rinse cycles that effectively selectively remove the aflatoxin from the initial sample solution. The aflatoxin, contained in the final rinse (elution) fluid, may then be placed in a fluorometer where its fluorescence can be measured and correlated to the level of aflatoxin in the initial sample.
Despite the existence of the above techniques, a need remains for a hand held, self-contained, automatic sensor with increased sensitivity to replace the less sensitive “wet chemistry” method and the large, cumbersome, chemical laboratory analysis systems. The new sensor should be viable for use not only against aflatoxin but against other analytes of agricultural, public health and defense interest as well. A modular design permitting the rapid substitution of different reagents and/or affinity columns to permit detection of various analytes with the same sensor would provide even greater benefits.
SUMMARY OF THE INVENTION
The sensor of the invention solves the above problems by offering the following important advantages: it is hand held, self-contained, automatic, low power, highly sensitive and selective, quantitative, stable with a long shelf-life, and fast (<2 min). It can be used against many different analytes with the reagents and/or affinity column appropriate for each analyte being inserted and removed as a single, modular unit.
The invention operates on the principles of immuno-affinity for specificity and fluorescence for a quantitative assay. It comprises two principal subsystems: a fluidic and chemical system that concentrates the analyte on an affinity column and elutes the concentrate into a small volume, and a sensitive fluorometer that measures the concentrated analyte's natural fluorescence. Both subsystems are located in the hand held, enclosed container that comprises the sensor of the invention. In one embodiment, the sensor can measure the concentration of aflatoxin in aqueous solution to concentrations down to 0.1 ppb.
After the sensor's built-in power supply is turned on, the user places a syringe with the sample solution in the sensor's external port and injects enough sample to clear bubbles through a bubble release valve. The operation of the sensor thereafter is entirely automatic, controlled by an on-board microcontroller. The intake of the sample and the clearing of bubbles through the bubble release valve could be automated as well.
The injected sample solution is drawn through an affinity column, which binds the analyte with great specificity. Next, a rinse fluid is drawn through the affinity column to wash it of any dissolved or suspended material that may later interfere with the fluorometric assay. The affinity column is, thus, washed clean, except for the chemically bound analyte. Next, a small, known quantity of elution fluid is drawn through the affinity column. This step releases the analyte, which is delivered in the elution fluid or eluant to a fluorometric cuvette.
The electro-optical subsystem or fluorometer is activated to measure the natural fluorescence of the analyte. A xenon arc lamp, run in single pulse mode, is used in the aflatoxin sensor embodiment of the invention as the radiation source. The arc lamp makes a flash on the order of a microsecond and radiation from the flash is captured by a first optical system or lens and is filtered by a UV filter to remove all light but a band in the near ultraviolet that excites aflatoxin fluorescence. The near ultraviolet light is then focused onto and transmitted through the fluorometric cuvette, and blue fluorescent light is emitted omnidirectionally by aflatoxin in the cuvette.
Some of the aflatoxin fluorescence leaving near 90° to the ultraviolet light path is captured by a second optical system and a second filter that passes only the blue fluorescent light emitted by the aflatoxin. The second optical system includes one or more lenses for focusing the fluorescent light from the cuvette on a detector, e.g., a photomultiplier tube (PMT).
The PMT, when illuminated by the blue fluorescent light, together with a the receiver circuit, measures aflatoxin based on the fluorescent energy from a single pulse of excitation. A transducer in the PMT produces on the order of a microsecond-long pulse of electrical current whose total charge is proportional to its light input, i.e., to the light generated by fluorescence in the sample, and, therefore, to the aflatoxin concentration.
In the receiver circuit, the PMT output drives a first operational amplifier circuit wired as a transimpedance amplifier with a low pass characteristic that is very long compared to the duration of the light pulse and the duration of the electromagnetic interference of the arc lamp. The transimpedance amplifier converts the charge (integral of the photocurrent) from the PMT into a decaying exponential pulse with amplitude and area both directly proportional to the charge from the PMT.
The output of the transimpedance amplifier is input to a track/hold circuit that is configured to make an output that tracks its input if an internal switch is in its normally closed condition. If the switch is placed in the open condition, the track/hold circuit holds the voltage at its output that was present immediately prior to the switch being opened. The track and hold circuit is switched into hold mode at the time that its output amplitude is the maximum in response to a pulse from the PMT.
The track/hold circuit reaches a peak response at a time long after a signal pulse comes from the PMT and the peak signal comes from the transimpedance amplifier. The track/hold circuit is placed in its hold mode also long after the pulse from the arc lamp occurred, and the track/hold circuit's output is directly proportional to the fluorescent light generated. Thus, the sensor output is decoupled from any electromagnetic interference generated by the sensor. While the hold condition is initiated at the time calculated for the peak signal, other times near that time would provide nearly equivalent results.
The track/hold circuit output/held value, i.e., the fluorescence intensity, is applied to the input to a digitizer
umerical display to be digitized and displayed to the operator of the instrument. Finally, after measurement is complete, the cuvette and plumbing are washed and backflushed and the system turns itself off.
If, unlike aflatoxin, the analyte to be detected does not have a measurable natural fluorescence, then a fluorescent tag can be added. For example, when the eluant leaves the affinity column, the eluant would be mixed with, depending on the analyte of interest, a fluores
Anderson Charles W.
Bargeron C. Brent
Benson Richard C.
Carlson Micah A.
Fraser Allan B.
Cooch Francis A.
Le Long V.
Pham Minh-Quan K.
The Johns Hopkins University
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