Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-07-15
2001-02-13
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120, C435S287100, C435S288700, C436S518000, C436S164000, C436S807000, C422S050000, C422S068100
Reexamination Certificate
active
06187530
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to an aquatic autosampler device. In particular, this invention relates to an autosampler device that permits collection of discrete water samples at many locations and depths for the purpose of quantifying and identifying micro-organisms at more frequent intervals than currently feasible.
BACKGROUND OF THE INVENTION
Understanding the presence, abundance, distribution, and population dynamics of micro-organisms that occur in aquatic environments requires frequent collection of discrete water samples at many locations and depths. Identification and enumeration of particular micro-organisms within those samples typically relies on laboratory-based methods that employ light and/or electron microscopy, and perhaps DNA, lectin, or antibody probes to reveal target species. Collecting appropriate samples over relatively large spatial and temporal scales (e.g., several square miles; months to years) is limited by the frequency one can spend visiting a particular location, and the absolute amount of time one can occupy that location. Similarly, providing quantitative measures of the abundance of a wide variety of micro-organisms that may inhabit those locations is restricted by the time and labor necessary for sample processing. In sharp contrast, many physical, chemical and gross biological properties of the water column may be determined in real-time using a variety of air borne, shipboard, moored and/or drifting sensor arrays. The disparity between the time required to gather and interpret physical and chemical measurements versus the effort to identify and enumerate particular micro-organisms in the same parcel of water, hampers the ability to generate synoptic views of the distribution and abundance of those species in an environmentally relevant context. This in turn impedes the ability to study, either in an applied or academic setting, a wide range of biological phenomena that is occurring at the single cell level, especially within a dynamic aqueous environment.
Molecular probe assays (DNA, PNA (peptic nucleic acid), lectin, or antibody) offer one means to speed and ease the detection and quantification of an enormous variety of organisms, as well the particular genes they harbor and express. However, such applications are presently hindered by the need for highly repetitive operations that demand trained personnel and specialized laboratory facilities. These requirements severely restrict the utilization of molecular probes for real-time ecological studies because the rate of sample processing is inherently limited and application of the technology outside of a laboratory setting is difficult, or more often impossible. U.S. Pat. No. 5,341,834, which is hereby incorporated by reference, discloses a multiport valve for a water transfer system for passing water drawn by a pump through multiple collectors. However, U.S. Pat. No. 5,341,834 discloses no information regarding the use of molecular probes for real-time detection of microorganisms in those samples.
Therefore, novel instrumentation is required if the analytical potential of molecular probe-based assays is to be merged in a synergistic fashion with existing and future capabilities of sensors that measure chemical and physical properties of aqueous environments. For environmental application, such an instrumentation package suitable for detection of micro-organisms should be portable, relatively simple to use, capable of autonomous operation in situ and have the capacity for real-time data transmission. To the best of our knowledge, instrumentation of this class does not exist.
SUMMARY OF THE INVENTION
A new class of instrumentation, an aquatic autosampler, was devised to meet the technological requirements outlined above. This instrument is designed to 1) collect discrete water samples of a known volume autonomously, 2) concentrate particles contained within those samples onto filter disks, 3) automate application of reagents such as preservatives, DNA, PNA, lectin, or antibody probes, and 4) identify and quantify particular species of micro-organisms so captured. Thus, this single electromechanical platform provides the necessary structure for collecting, storing, and processing samples using any one of a number of formats (whole cell or cell homogenate) or molecular probe techniques (DNA, lectin, antibody). The aquatic autosampler collects samples and can process the samples in a variety of ways. The autosampler can process whole cells or cell homogenates and can use different kinds of probes that are either attached to the solid supports, free in solution, or a combination thereof.
The aquatic autosampler has the following major components: a syringe sub-assembly, valve manifolds, reagent bags, a filter carousel having carousel tubes, a filter shuttle, and an imaging system. In operation, a filter disk enclosed within a filter housing is driven up a carousel tube and into the filter shuttle. The shuttle then moves the filter housing into the “process” position, where the sampling takes place. When the filter housing is in the “process” position, the syringe inhales water or other fluid through a valve manifold. The fluid passes through the filter housing and the filter disk, which collects a discrete sample. A toggle valve on the syringe sub-assembly allows the syringe to discharge the fluid, without passing the fluid through the filter a second time, by discharging the fluid through another valve manifold.
Reagents, such as preservatives, detection solutions or molecular probes, contained in the reagent bags may then be drawn across the sample collected on the filter disk, using the syringe. The molecular probes, such as DNA, lectin, and antibody probes, help identify the particular organisms that are present in the sample. The filter shuttle then moves the processed filter to the “unload” position, where an image may be acquired for real-time detection of micro-organisms or, alternatively, the filter housing is sent down a carousel tube for storage and later analysis.
The aquatic autosampler can function inside of as well as outside of a laboratory, and can be moored for autonomous function submerged within the water column when packaged inside a suitable pressure housing. The aquatic autosampler will record results of preprogrammed analytical functions and/or relay that data to a remote location immediately following a process.
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Brown Mark
Cline Danelle E.
Massion Eugene I.
Mellinger Ed
Scholin Christopher A.
Chin Christopher L.
Limbach & Limbach LLP
Monterey Bay Aquarium Research Institute
Pham Minh-Quan K.
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