Bioassays sensitivity

In the past few years our research laboratory has been working on the applications of a new technique, called microwave-accelerated metal-enhanced fluorescence (MAMEF), as applied to fluorescence-based bioassays. The MAMEF technique couples the benefits of low power microwave heating witii metal-enhanced fluorescence (MEF), to address the two major shortcomings of fluorescence-based bioassays currently in use today i.e., bioassay sensitivity and rapidity. In MAMEF, [1] the MEF phenomenon increases the sensitivity of the assays, while the use of low power microwave heating kinetically accelerates assays to completion within only a few seconds. 

Maximum acceptable concentiation figures expressed as fractions of 96 h for most sensitive species in given area. The 96 h is that concentration of a substance which kills 50% of the test species within 96 h under standard bioassay conditions. 

Bioassays are based on the growth response of vitamin-depleted rats or chicks to graded amounts of vitamin B 2 added in the diet. These assays are not specific for vitamin B 2 because factors, other than vitamin B 2 present in biological materials, produce a growth response. Because coen2yme primary form of natural vitamin 2 is light sensitive, assays should be carried out in subdued light. 

It is obvious from the provisional risk assessment values for microcystins, and, being of the same order of magnitude of mammalian toxicity, similar values may be calculated for the cyanobacterial neurotoxins, that sensitive detection methods are required to detect these low concentrations of toxins. Of the biological methods of detection discussed earlier, the mouse and invertebrate bioassays are not sensitive enough without concentration of water samples, in that they are only able to detect mg of microcystins per litre. Only the immunoassays (ng-/rg 1 and the protein phosphatase inhibition assays (ng O... 

The mysid shrimp, Mysidopsis bahia, is the test organism for the liquid and suspended particulate phases. This species has been shown to be exceptionally sensitive to toxic substances and is considered to be a representative marine organism for bioassay testing by EPA. An LCj, is determined the suspended particulate phase (SPP) bioassay tests. 

The mouse bioassay for PSP, described in its original form by Sommer in 1937 (29), involves i.p. injection of a test solution, typically 1 mL, into a mouse weighing 17-23 g, and observing the time from injection to death. From the death time and mouse weight, the number of mouse units is obtained by reference to a standard table 1 mouse unit is defined as the amount of toxin that will kill a 20-g mouse in 15 min (77). The sensitivity of the mouse population used is calibrated using reference standard saxitoxin (70). In practice, the concentration of the test solution is adjusted to result in death times of approximately 6 min. Once the correct dilution has been established, 5 mice will generally provide a result differing by less than 20% from the true value at the 95% confidence level. The use of this method for the various saxitoxins and indeterminate mixtures of them would appear... 

Until the recent development of appropriate HPLC techniques capable of detecting pmol amounts (see Flentge et al. 1997) ACh could only be measured chemically by relatively lengthy and expensive procedures (e.g. gas chromotography), which were not always very sensitive, or by bioassays. Although the latter, using muscle preparations that responded to ACh, such as the dorsal muscle of the leech, the rectus abdominus of the frog or certain clam hearts, were reasonably sensitive they were tiresome and not easily mastered. Thus studies on the release and turnover of ACh have not been as easy as for the monoamines. 

Bioassay of alternate molecular forms supports the view that the ORs are capable of resolving isomeric distinctions in neutral (non-biological) odourants. Stereochemical pairs of odours were tested for differential sensitivities in the blind subterranean mole rat (Spalax ehrenbergi). The subjects responded to one enantiomer, but not to its stereoisomer. Both sexes were attracted to the odour of R-(-)-carvone but unresponsive to S-(+)-carvone in contrast, males and females were repelled by the odour of (+)-citronellol, but not by (-)-citronellol (Heth et al., 1992). The lack of responsiveness by mole rats could be central due to lack of salience, or peripheral due to hyposmia/anosmia for one isomer. Both carvones have distinct odours for the human nose. 

Other kinds of bloassays have been used to detect the presence of specific allelochemical effects (8), effects on N2 fIxatlon (9), the presence of volatile compounds (10) and of Inhibitory substances produced by marine microalgae (11). Putnam and Duke (12) have summarized the extraction techniques and bioassay methods used In allelopathy research. Recent developments In high performance liquid chromatography (HPLC) separation of allelochemlcals from plant extracts dictates the need for bloassays with sensitivity to low concentrations of compounds contained In small volumes of eluent. Einhellig at al. (13) described a bloassay using Lemna minor L. growing In tissue culture cluster dish wells that maximizes sensitivity and minimizes sample requirements. 

Additional research with Lemna species has indicated that parameters other than direct growth measurements may be more sensitive In this bioassay. Table VII compares growth and chlorophyll content of L. minor as affected by catechln. Final frond number was inhibited by 1000 pM catechln and stimulated at lower concentrations of 50 and 100 pM. Chlorophyll content on a per-frond basis, however, was consistently Inhibited by catechln and was concentration dependent to 100 pM. 

A bioassay which is generally accepted in the discipline as representative and sensitive for the biological effect. 

The objective of the research reported here was to develop sensitive bioassays which utilize near-whole plant systems of appropriate target aquatic weeds and which require little space and low volumes of incubation medium. Such bioassays could be used to help identify active fractions of chromatographically partitioned allelochemicals and could also be used in prinary screening procedures for newly synthesized agrichemicals. 

Ultimately, it is important to develop explant bioassays which can help quantify allelochemicals. This will require greater availability of purified active products. However, at this time, the explant systems can be used to show qualitative effects whether in crude extracts, leachates or from HPLC fractionation (17). Once target species effects are characterized, other bioassays which may be more sensitive could be used as well. 

J. Chayen, Cytochemical bioassay and its potential place in compendial definitions A method that offers sensitivity as well as specificity, in Hormone Drugs, U.S. Pharmacopeial Convention, Rockville, MD, 1982, pp. 48-58. 

The photophysical properties of lanthanide ions are influenced by their local environment, the nature of the quenching pathways available to the excited states of sensitizing chromophores, and the presence of any available quenchers (as we have seen when discussing bioassay). All of these factors can be exploited for the sensing of external species. 

The main advantage of this technique is the rapidity of response and the possibility of performing many bioassays with different algal strains at the same time. The disadvantage is that the kind of response is qualitative. This limitation can be overcome by selecting for liquid inhibition bioassays, the strains that give a positive response in paper disk bioassay (i.e. the strains sensitive to the allechemical(s) tested). 

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