FIELD OF INVENTION
This invention pertains to the chemical and bio-molecular testing and control of cellular behavior to characterize composition and production of chemical species from biological activities, particularly those activities pertaining to aquatic or marine ecosystems.
BACKGROUND OF THE INVENTION
The primary method for studying marine ecosystems for evaluation of chemical ecology is to harvest and sacrifice the biological species before analysis. Although these techniques (we term blender chemistry) provide rich chemical information from biological systems, they lose information in the process by stressing the sampled organisms and potentially changing measured components. In addition, sampling techniques prior to this fail to provide adequate spatial and temporal information to capture communication between species via semiochemical release (chemical ecology). This requires much higher spatial resolution, temporal resolution, and feedback with the organism in a viable ecosystem while sampling to ensure the environmental or behavioral conditions haven't changed. Blender chemistry simply provides an integration of sample components and loses much of the valuable temporal and spatial components of biological behavior. In addition, significant sensitivity enhancement is required to measure many of the trace metabolites and semiochemicals that end up diluted when using blender methods. The prior technologies fail to sample in time windows that capture measurable amounts of sample before being diluted into an essentially infinite reservoir of liquid (e.g. the ocean).
DESCRIPTION OF FIGURES
FIG. 1—Table indicating selected Interaction Parameters, Target Organisms, Sample Introduction Means, and Monitoring Means for use with the current invention. The invention is intended to allow spatial and temporal measurements of the interaction of biotic and abiotic components of a living ecosystem, whereby the chemical interaction, products of metabolism, and behavior of the organism can be monitored and measured. The items on this list are intended to represent examples of measurement parameters but are not intended to limit the application of the present invention in scope.
FIG. 2—Flow diagram of the major components of a aquatic sampling system. These components comprise a plurality of sampling probes labeled A through D, a sample positioning means for adjusting the position of sample probes relative to the sampled organism, a sample collection means to control the sampling time and volume (in this embodiment a peristaltic pump with multiple sampling streams), a sample conditioning means (filters, salt washes, addition of buffers), a sample enrichment means (this case a solid phase extraction means to isolate and enrich sample components), a sample analysis means (in this case a liquid chromatographic column with gradient separation conditions interfaced to a mass spectrometer). Also required for effective operation of the sampling system is a variety of switching and control devices to facilitate the discrete processes of sampling, enriching, conditioning, separating and analyzing components.
FIG. 3—Schematic diagram of a marine organism showing a tentacle sampling probe designed to milk the tentacles for a wide variety of chemical components or chemical signature of biological condition or response to stimuli. The sampling probe is observed with an external monitoring means and the sampling probe is able to assume an optimal position relative to a moving physical feature of the sampled organism.
FIG. 4—A photograph of a simple tubular sample probe (600 um) performing non-contact sampling of a micro-feature (polyp) of an acropora. Note the collection of respiratory gases from the organism on the sample probe tip.
FIG. 5—A photograph of a cultured reef environment with an extended microscopic video camera (in this case with an 18 inch focal length) used to monitor, position, and adjust the position of sampling probes in order to maintain consistent sampling distance, or potentially to protect the organism harm or damage from closely positioned sampling probes. Shown in the photograph are the sample tank containing acropora and other species, a four probe sample positioning apparatus, and a four line peristaltic sampling pump.
FIG. 6—Embodiments of non-contact sampling probe geometries intended for various application and types of interactions with sample organisms or sample surfaces; showing a) a simple tubular probe for withdrawing liquid directly from sample regions, b) an angled tubular probe for addressing organism geometries that are more complex or inaccessible with linear probes, c) a fritted or porous tipped probe to collect sample through a higher collection cross-section while preventing particulate material from entering the sampling stream, and d) a blank tipped probe with radial sampling holes oriented around the region near the tip of the probe.
FIG. 7—Embodiments of contact sampling probes intended to interrogate the chemical composition of surface attached sample components at various organism surfaces or physical features; showing a) a swab tipped probe intended to make contact with the sample organism without damage to the surface of the organism, b) a conical tipped probe made of either soft or hard material with radial holes intended to sample a radius of surface defined by the tip geometry from the sample organism, c) a conical tipped probe similar to b) without radial holes, said conical tipped probe conducting liquid to the sample to sweep the surface and collecting sample containing liquid through a coaxial outlet tube as indicated by the arrows, and d) a brush tipped (soft or hard brush) to agitate sample surface to dislodge sample material for subsequent collection through the outlet of the tube.
FIG. 8—Embodiments of micro-sampling probes that are intended to extract small volumes of sample at or near the surface of an organism, showing a) a microtube mounted in the tip of a larger transfer tube, b) a microtube mounted in the tip of a larger transfer tube with input or output flow paths for introducing sample collecting liquids to the sample probe and collecting small volumes of sample through the microtube into the higher flow input and output steams, and c) a microtube mounted to a microsyringe to extract discrete sample volumes of sample local the organisms utilizes a micro-switching valve to switch from loading to injection positions.
FIG. 9—Embodiments of alternate sampling probes that are intended to selectively extract volumes of sample, showing a) a dielectric tube containing sampling probe sheath by a grounded cover, said dielectric tube having an attractive voltage applied a remote end of the dielectric tube in order to attract ions to and through the tube with a continues field along the length of the dielectric, b) an electrically biased tube insulated from an electrically grounded outer sheath that attracts oppositely charged sample ions to the said biased tube and said charged sample ions are swept into the tube and collected, c) a chemically modified sample probe that allows for the attachment of functional groups that show affinity for selected sample components or provide stimuli to the organism and d) a specialized sampling tip for insertion of sampling tip into sediment to a controlled or metered depth to interrogate species that are associated with subsurface organisms.
DESCRIPTION OF INVENTION
The current invention describes devices and methods intended to monitor (and additionally) measure the behavior of selected in vivo or in vitro aquatic organisms; including, plants, animal, and micro-organisms. The Table presented in FIG. 1 is intended to itemize examples of the types of behavior and environmental conditions that serve as parameters of interest in studying marine ecosystems. The device is intended to measure species specific biomolecules as a function of experimental conditions during in vitro experiments, or species specific biomolecules as function environmental conditions during in vivo experiments. The present invention allows the measurement of biochemical products of biological activity. Monitoring the interaction of living organisms requires the measurement of both biotic and abiotic parameters within the observation regions.
The invention comprises one or more methods for direct sampling in vivo or in vitro marine ecosystems that comprise:
- a sample or plurality of samples,
- a means for sampling,
- a means for collecting samples,
- a means for monitoring sample organism for position and behavior to optimize
- sampling position and time,
- a means for positioning said sampling means,
- a means for conditioning sample,
- a means for enriching sample components,
- a means for analyzing said sample components,
whereby the sampling of the said sample is controlled both spatially and temporally in by in order to characterize and relate chemical and physical behavior of biological organisms within a given ecosystem. A representative number of sample organisms, interactions from the ecosystem, sample introduction means, and monitoring means are itemized in FIG. 1. It is a primary objective of the present invention to monitor the biotic and abiotic interactions at the organism, organism sub-feature, or cellular level. Monitoring the experimental conditions for the devise may include one or more video, chemical, optical, or physical measurement in order to correlate behavior relating to experimental or environmental conditions. Cultured ecosystems have an advantage of allowing the experimenter to more fully control the environmental conditions and composition, both abiotic and biotic.
Preferred Embodiment
A preferred embodiment is schematically illustrated in FIG. 2. The cultured marine ecosystem is schematically represented as an aquarium tank containing species A and B. Four sample probes are inserted into the tank at various observation positions relative to the observed species. One probe at organism A, one at organism B, one at the interface between the two species, and one in the background as a control measurement. Each sample probe (A through D) can be manually or automatically positioned by a sample positioning means. The temporal and spatial position of each sample probe can be fixed or movable depending on the nature of the experiment, one alternative embodiment of this invention used a monitoring means to observe the conditions of both sample and observed organism. For example, the monitoring means are capable of monitoring movement of the observed species and automatically changing sample probe position relative to sample organism. All four probes can be discretely and/or simultaneously sampled by a peristaltic pump which serves as a collection means. The sample streams are collected and delivered for analysis. A wide variety of sample conditioning means, sample enrichment means, and sample analysis means are integrated into the system for sample pre-treatment, filtering, and pH adjustment. The conditioned sampling lines are each delivered to a solid phase extraction column where sample can be enriched, and desalted. The sample enrichment means of SPE provided for enriched sample to be delivered to an analysis means comprising liquid chromatography mass spectrometry.
This embodiment utilizes a number for switch valve in the conditioning, enrichment, and analysis phases of analysis to enable sample loading, unloading, elution, and regeneration with clean solvents to prevent carryover between samples. Valve also allow switch from sample to sample stream, and sample to sample time (same sample stream, different time). Sample volumes and sampling durations can be varied to accommodate sampling requirements.
This invention utilizes monitoring means for measuring one or more of the following attributes of the sample ecosystem;
- a) monitoring the position and spacing of sampling probes relative to sampled organism or region,
- b) monitoring the visual appearance of organisms in various states of health or under various states of behavior (e.g. feeding, preying, reproducing, defending),
- c) monitoring a organism specific response such as fluorescence or emission,
- d) monitoring the background levels or populations of biotic materials (e.g. algae, plankton)
- e) monitoring the background levels abiotic materials (e.g. salts, hydronium ions)
- f) monitoring the abiotic parameters (temperature, current, temperature gradients, turbidity).
Monitoring means are intended to be used for the following objectives;
- a) feedback information on position or positions of samples relative to sampling probes,
- b) feedback information to correlate biochemical results related to monitored parameters,
- c) feedback information to correlate biochemical response induced stimuli.
Additional Preferred Embodiments
Non-Contact Sampling
FIG. 3 illustrates the relationship between sample probe and physical feature of the target organism. This figure shows a tubular sampling probe positioned out the apex of a tentacle in order to collect chemical output from the region of the acrosphere. A monitoring means is used to align the sampling probe with the tentacle. The device is capable of manual or automated alignment. FIG. 4 shows a photograph of sampling probe interaction with an aquatic species of hard coral called acropora. The acropora structure sampled in this experiment was the polyp. Exact positioning of the sampling probe is important to capture species specific molecules associated with the oral grove at the end of the polyp. The probe dimension in this photograph is 600 um diameter. Small species features can be matched with small dimensioned and/or geometrically appropriate probes. FIG. 5 is a photograph of the monitoring means for the present invention whereby organism can be observed with a long focal length (18 inch) microscope that is movable and focusable relative to selected sample species in the tank. A multiple sampling probe holding and positioning assembly is locate above the tank allowing discrete positioning of sample probes relative to sample organisms. Manual or automated control of both monitoring microscope and sample probes is possible.
Embodiments of non-contact sampling probe geometries intended for various applications and types of interactions with sample organisms or sample surfaces are shown in FIG. 6. The example non-contact sampling probes are show as, a) a simple tubular probe for withdrawing liquid directing from sample regions, b) an angled tubular probe for addressing organism geometries that are more complex or inaccessible with linear probes, c) a fritted or porous tipped probe to collect sample through a higher collection cross-section while preventing particulate material from entering the sampling stream, and d) a blank tipped probe with radial sampling holes oriented around the region near the tip of the probe.
These non-contact probes have the advantage of sampling aqueous environments in marine ecosystems without contact in potentially stimulating chemical response from sample organisms. These have particular utility for evaluating extra-cellular materials for defense response, attractant emission, repellent emission, and products of metabolism.
Additional Preferred Embodiments
Contact Sampling
FIG. 7 illustrate a number of embodiments of contact sampling probe geometries intended for various applications and types of interactions with sample organisms or sample surfaces. These embodiments of contact sampling probes are intended to interrogate the chemical composition of surface attached sample components at various organism surfaces or physical features; showing a) a swab tipped probe intended to make contact with the sample organism without damage to the surface of the organism, b) a conical tipped probe made of either soft or hard material with radial holes intended to sample a radius of surface defined by the tip geometry from the sample organism, c) a conical tipped probe similar to c) without radial holes, sail conical tipped probe conducting liquid to the sample to sweep the surface and collecting sample containing liquid through a coaxial outlet tube as indicated by the arrows, and d) a brush tipped (soft or hard brush) to agitate sample surface to dislodge sample material for subsequent collection through the outlet of the tube.
FIGS. 7
a-c are intended to dislodge sample components from the surface and sweep said dislodged sample components into the outlet of the respective sampling tube. The swab-type probe is intended to prevent damage to the surface of the organism while allowing collection of components that are not dissolved in the surrounding ambient water (bulk fluid). Probe 7b and 7c with conical tip allow pseudo sealing of the surface around the cone with the edge of the cone. In FIG. 7b, the ambient water is drawn into the sampling region (inside the cone) to sweep analytes off the surface through simple turbulence. In the case of FIG. 7c, liquid input from the sampling probe is directed at the organism surface and subsequently drawn to the outlet tube for collection and analysis. The sampling cone is made of either soft (flexible) materials or hard depending on the properties of the organism surface being sampled.
One alternative embodiment of FIG. 7c would entail the addition of reagent solutions that are intended to stimulate or react in some way with the sampled organism to induce the output of a chemical response. Products of that response are drawn to the sampling tube and analyzed.
FIG. 7
d utilizes the bristles of a brush to agitate components on the sample surface in order to dislodge and entrain components in the liquid flow toward the outlet tube. The bristles are intended to be either soft for sensitive surfaces or hard for dislodging strongly bound components of durable surfaces. This technique would be good for collecting micro-organisms as well as chemical species. We envision condition micro-organisms collected from surface or suspended into the ambient water to require special pre-treatment such as cell lysing and isolation of cellular fractions, before chemical analysis.
Additional Preferred Embodiments
Small Volume of Micro-Sampling
FIG. 8 illustrates a number of embodiments of the sampling probe for sample volumes that are much smaller (100 uL to sub-uL). Many applications of ecosystem analysis require significantly enhanced spatial arnd temporal resolution to measure bioemmission in short time domains for from small physical regions of the organisms. The present sampling probe embodiments include designs for reduced size tips and reduced volume sampling in order to increase both spatial and temporal resolution. Embodiments of a micro-sampling probes that are intended to extract small volumes of sample at or near the surface of an organism are shown. These include a) a microtube mounted in the tip of a larger transfer tube, b) a microtube mounted in the tip of a larger transfer tube with input or output flow paths for introducing sample collecting liquids to the sample probe and collecting small volumes of sample through the microtube into the higher flow input and output steams, and c) a microtube mounted to a microsyringe to extract discrete sample volumes of sample local the organisms utilizes a micro-switching valve to switch from loading to injection positions.
Discrete small volume sampling requires both in FIG. 8c allow small volumes to be collected into a sample loop, injected into a higher flow stream for rapid transmission away from the sampling environment for pseudo-realtime analysis.
Additional Preferred Embodiments
Specialized Sampling Probes
FIG. 9 illustrates a number of embodiments of the sampling probe for collecting specific chemical components from the sampled organism or organisms. Embodiments of specialized sampling probes are intended to selectively extract volumes of sample. Embodiments are shown in FIGS. 9a-d, showing a) a dielectric tube containing sampling probe sheath by a grounded cover, said dielectric tube having an attractive voltage applied a remote end of the dielectric tube in order to attract ions to and through the tube with a continues field along the length of the dielectric, b) an electrically biased tube insulated from an electrically grounded outer sheath that attracts oppositely charged sample ions to the said biased tube and said charged sample ions are swept into the tube and collected, c) a chemically modified sample probe that allows for the attachment of functional groups that show affinity for selected sample components, and d) a specialized sampling tip for insertion of sampling tip into sediment to a controlled or metered depth to interrogate species that are associated with subsurface organisms.
Specialized probes are intended to enhance to collection efficiency of selected components, generally at the expense of other components.
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Patent Application
20140349328
