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Single molecule detection of target molecules specifically bound by paired fluorescently labeled probes has shown great potential for sensitive quantitation of biomolecules. To date, no reports have rigorously evaluated the analytical capabilities of a single molecule detection platform employing this dual-probe approach or the performance of its data analysis methodology. In this paper, we describe a rapid, automated, and sensitive multicolor single molecule detection apparatus and a novel extension of coincident event counting based on detection of fluorescent probes. The approach estimates the number of dual-labeled molecules of interest from the total number of coincident fluorescent events observed by correcting for unbound probes that randomly pass through the interrogation zone simultaneously. Event counting was evaluated on three combinations of distinct fluorescence channels and was demonstrated to outperform conventional spatial cross-correlation in generating a wider linear dynamic response to target molecules. Furthermore, this approach succeeded in detecting subpicomolar concentrations of a model RNA target to which fluorescently labeled oligonucleotide probes were hybridized in a complex background of RNA. These results illustrate that the fluorescent event counting approach described represents a general tool for rapid sensitive quantitative analysis of any sample analyte, including nucleic acids and proteins, for which pairs of specific probes can be developed.

In the context of proteomic research, affinity separations for the prefractionation of complex mixtures, such as cell lysates or human tissues, have become increasingly important. Microfluidic devices have shown significant potential to achieve fast analysis and low sample consumption. Here, we demonstrate the use of a microfluidic device to achieve affinity capture of albumin from human cerebrospinal fluid. Traditional photolithography and wet etching techniques were used to fabricate devices from borosilicate glass wafers. Monolithic porous polymer was prepared in a microfluidic channel by photopolymerization of glycidyl methacrylate and trimethylolpropane trimethacrylate. After derivatization with Cibacron-blue-3G-A, the modified polymer was used to achieve affinity capture of lysozyme and human albumin. Both fluorescence detection and matrix-assisted laser desorption ionization time of flight mass spectrometry were used to validate the results.

We describe the performance of a new glass attachment chemistry for arrays that is particularly well suited to attachment of small molecules, such as peptides. The attachment chemistry is a protected isocyanate (PI) group. Isocyanate groups are well suited to serving as a glass coating for arrays, in that they are highly reactive with many different types of biological compounds. However, they are generally so reactive as to be unstable. The new feature of the PI slide coating is its stability. It can withstand immersion in water without loss of reactivity and has at least a 1-year shelf life. The high reactivity of the PI group results in a rapid coupling reaction (< 15min) and is particularly useful for attaching small molecules, such as peptides. Since isocyanates bind to both amines (forming a urea linkage) and hydroxyl groups (forming a carbamate bond), we tested the ability of the PI coating to bind to a wide variety of compounds. We found that the PI slide coating can directly attach to peptides, proteins, carbohydrates, lipooligosaccharides, and DNA. The sensitivity of detection for these compounds is comparable to that of other previously published array substrates.

In this study, we compared the efficiency of capture DNA probes covalently bound onto magnetic beads or microplates for their hybridization with target human cytomegalovirus (HCMV) DNA amplicons. Polystyrene supports were first aminated by wet chemistry to allow covalent grafting of the capture probes. The level of amines grafted was three times higher on beads than on microwells. Increasingly higher sizes of capture probes were fixed on both supports and the best reaction yield ranged from 300 to 500 fmol. The sizes of the capture and detection probes were optimized in order to obtain high target DNA hybridization yield. Long capture probes were more accessible than short ones to the target, with faster kinetics of hybridization obtained on beads than on microplates. Sensitivity of the hybridization assay was then determined with a nonisotopic method and the detection limit found was 30 amol of HCMV amplicons on both supports. HCMV DNA extracted from clinical samples were amplified by PCR. The resulting amplicons were then analyzed using the optimized sandwich hybridization assay discussed here. The results perfectly fitted with the qualitative conclusions obtained after a nested PCR analyzed on agarose gel., (Copyright 1997 Academic Press.)

The identification of chemical species and the measurement of their concentrations with high (submicrometer) spatial resolution are of considerable importance in cell biology. In this article we report the first successful development of a > or = 0.1-micron Ca2+ sensor based on a pulled micropipet, filled with a conducting porous sol-gel glass which was doped with the fluorescent calcium green 1 Ca2+ indicator. Such sensors are potentially capable of measuring Ca2+ concentrations as low as 10(-8) M, in confined volumes, with a three-dimensional resolution which exceeds approximately 0.1 micron. A major advantage of the sensor is its capability to be integrated into a multifunctional probe which will measure chemical analyte concentrations and ion conductance.

Individually wrapped, sterile disposable transfer pipets can be used in the isolation of ds-DNA and ds-RNA fragments from gels as well as in the screening of multiple samples in Southern, Northern, and Western blots without potential contamination by exogenous nucleases and proteases. The sensitivity and results obtained by this method are comparable to those obtained by conventional methods. All the prehybridization, blocking, hybridization, and detection processes can be performed within the transfer pipet. The isotopically labeled probes used in hybridization can easily be recovered, stored for reuse, or disposed of as waste with no potential contamination of personnel or laboratory equipment. Strip blots are stable in appropriate buffers within the liquepipets which can be shipped easily worldwide for comparative analyses by collaborative investigators. This method is simple, time saving, and inexpensive and is particularly suitable for multiple sample screening. Other potential applications of this procedure are discussed.