There are many different techniques currently available to screen biological samples or collections, and mutant or gene-expression libraries for those individuals that have distinguishing or desired characteristics. Most procedures require individual colonies that are grown from the samples, collections or libraries to be picked from Petri dish into microtiter dish format, then incubated for a period of time to allow growth, followed by considerable sample handling and preparation, all of which is prior to the analytical step. When many individuals must be analyzed, these steps are undesirable. Methods of analysis that require less sample preparation have been developed for use in screening large numbers of samples.
An improvement to mass spectrometry sample screening methods allowing high throughput assay of many samples was described in WO 00/48004. This method eliminates the column separation step that is generally required prior to sample injection into the mass spectrometer by purifying components of interest using processes such as adding a volatile buffer, an organic solvent, or an ion exchange resin. In addition, components could be purified by attachment to a solid support. However, this method still requires that prior to the analysis, some purification of component(s) of interest is performed, and the purified sample is then injected into a mass spectrometer.
Elimination of sample component purification in a screening method is described in U.S. Pat. No. 5,914,245. In this method, microcolonies of cells on a base are optically monitored over time for changes in an optically detectable signal. The optically detectable signal arises from contacting the cells with an optical signal substrate, from which the optically detectable signal is produced through an enzymatic reaction. The monitoring of the optically detectable signal over time allows for characterization of the activity of the enzyme produced by the microcolony. In this method the cells may be lysed or permeabilized to expose the optical signal substrate to the cellular contents. Though this method does eliminate sample purification steps prior to analysis, its limitation is that the analysis is based on optical detection, which makes the method applicable only in cases where optical substrates with appropriate absorbance or fluorescence properties are available for the enzyme to be assayed.
In U.S. Pat. No. 6,472,163, the method of U.S. Pat. No. 5,914,245 is improved through the use of different types of microporous membranes, which are easy to handle and have chemical resistance, as the base. Additional improvements include better temperature control, more compact illumination systems, and the use of different indicators for direct and indirect assays or coupled assays. The limitation remains that all assays are monitored by absorbance or fluorescence techniques.
Detection of organic molecules using Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) in a high-density array sample format is described in Braun et al. (Anal. Chem. (1999) 71: 3318-3324). In this process samples are injected into silicon nanovials, which provides for analysis of femtoliter to picoliter sample volumes with compound concentrations of ×10−2 to 1×10−4 molar allowing detection of attomole to femtomole quantities of molecules. This approach is required when the only means of mapping is via rastering of the primary ion beam, which limits the field of view to less than 1000×1000 μm2. Thus the extremely sensitive capacity of the ToF-SIMS detection was applied in a high throughput miniaturized assay system, however only solutions containing a pure compound were assayed. Thus this assay system, as described, is applicable to simple solutions that require transfer of femtoliters into an array of chambers prior to analysis. Extension to screening of microbial products still requires culturing time and considerable sample handling to transfer large numbers of complex and/or filtered solutions to the array.
The TOF-SIMS assay technique has been used previously in the analysis of many types of biological samples, all of which have required sample preparation procedures prior to placement of the sample in the ToF-SIMS instrument. Types of biological samples that have been analyzed by TOF-SIMS include peptides prepared from purified proteins (Jabs and Assmann; Journal of Chromatography (1987) 414(2): 323-333), protein or other cellular components binding to metal surfaces (Michel et al. Langmuir (2002) 18(8):3281-3287; Sjoevall et al. Analytical Chemistry (2003) 75(14): 3429-3434), microbial cell walls in freeze-dried preparations (Tyler et al, Proceedings of the International Conference on Secondary Ion Mass Spectrometry, 12th, (2000) pp 943-946, Editors: Benninghoven, Alfred. Publisher: Elsevier Science B.V., Amsterdam, Neth.), model phospholipid membranes following freeze-fracture (Cannon et al. Journal of the American Chemical Society (2000), 122(4): 603-610), freeze-fractured and frozen-hydrated liposomes and red blood cells (Pacholski et al., Rapid Communications in Mass Spectrometry (1998), 12(18:1232-1235), freeze-fractured and frozen hydrated paramecium (Colliyer et al., Analytical Chemistry (1997), 69(13): 2225-2231), and radioactive isotope labeled compounds in fractionated cells (Glassgen et al., Pesticide Biochemistry and Physiology (1999), 63(2): 97-113).
All of the methodologies and biological sample types described above have limitations for screening of large numbers of samples to identify the rare, desired sample due to sample handling and/or detection processes. The problem to be solved, therefore, is to develop a methodology that would permit direct analysis of individual organisms without sample handling and preparation and without restriction to absorbance and fluorescence techniques.
Applicants have solved the stated problem by developing a rapid and efficient method of applying Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) to the analysis of intact microorganisms. Using this method, biological products of organisms that are intact upon introduction into the ToF-SIMS instrument are screened by surface analysis to allow characterization of the individual organism. The individuals are grown on, or transferred to, the surface of a membrane such that they form an array. Further, the array of intact organisms may be processed in situ and directly introduced into the ToF-SIMS instrument. Use of this method allows rapid screening of arrays of mixed populations of microorganisms such that individuals having different cell contents are identified. Therefore this method provides a substantial advancement over available methods of screening large biological collections to identify individuals of interest.