Chemiluminescence in Analytical Chemistry

Figure 4 Quartz microbalance electrode with a protein A-HRP conjugate immobilized on the gold surface. (a) Transmitted light image; (b) chemiluminescent signal after addition of CL substrate; (c) 3-D display of the light signal spatial distribution.

and the light signal was obtained after the addition of a CL substrate for HRP (Fig. 4b). Figure 4c shows the pseudocolor-processed CL image and the 3Ddisplay of the light signal, which allow a better evaluation of the immobilized protein distribution.

5. CHEMILUMINESCENT ANALYSIS OF MICROSAMPLES

The analytical performance of CL microscopy imaging in terms of detectability, precision, accuracy, and spatial resolution was previously reported by us [25]. The system allowed for the detection of 400 amol of enzymes such as HRP or AP, with a spatial resolution as low as 1 m and very low background. CL coupled with optical microscopy thus represents a useful tool for enzyme, antigen,

and DNA probe localization [29, 30] and is particularly suitable for those samples that require high detectability and, at the same time, quantitative information. This method can be applied to any kind of specimen such as fixed cells, tissue cryosections, and paraffin-embedded sections. The specimens are prepared following the same protocols developed for enzymatic, immunohistochemical, and in situ hybridization techniques with other detection systems. The resolving power of the CL image obtained using a videocamera connected to an optical microscope is comparable to that obtained with the optical microscope and a transmitted light image, making it possible to localize a CL signal in a tissue section or within a single cell, which is particularly suitable for analysis at the subcellular level [31–33, 35, 58].

CL mapping of the analytes in the specimen requires three steps: first, transmitted light image collection; second, addition of the CL cocktail and acquisition of the emitted light; third, after computer processing of the luminescent signal with pseudocolors corresponding to the light intensity, superimposition of the CL image with the live image to correctly localize the analyte. The multistep procedure can be facilitated by the use of an optical microscope equipped with a computer-assisted device able to precisely locate the same sample spots. With an automated system the detection of two or more reactions in the same specimen can also be easily performed, thus increasing the diagnostic power of such techniques.

Semiquantitative analysis can be performed once the system is optimized taking into account the nonspecific chemical and electronic background signal [25, 59]. Since quantification could be imprecise or inaccurate owing to factors such as sample shape, light-scattering phenomena, and internal reflectance in the reaction medium, negative and positive control experiments with the same type of specimens under the same experimental conditions must be performed and the results expressed as a relative value [47]. Therefore, a positive signal in CL microscopy imaging is generally considered to be above a predetermined threshold value (usually, the background value plus five standard deviations). An objective evaluation of the results can be achieved without any microscopic training thus minimizing uncertainties about positive or negative results. When tissue sections are analyzed, their thickness must be as reproducible as possible because there is a linear relationship between light emission and section thickness [35]. The analyte concentration in cell or tissue samples could be quantified using a calibration curve obtained by immobilizing different amounts of analyte on a target surface such as activated oxirane acrylic beads or nylon net, as previously reported [25, 36].

5.1 Enzyme Activity

Endogenous AP activity was detected in rabbit intestine cryosections by simply adding a few drops of CL substrate [25]. AP was localized in the epithelial cells

with good resolution and sensitivity, the aspecific signal being very low (Fig. 5a). Diffusion of the chemiluminescent products was minimized by removing excess solution from over the cryosection, after which the chemiluminescent substrate was still in excess and embedded in the tissue. This method is superior to conventional histochemical colorimetric detection of AP, since experiments performed on the same specimen, first analyzed with CL and then by a conventional colorimetric technique, showed that CL is more sensitive and also permits a more accurate quantification of the enzyme [60].

Endogenous AChE activity was detected in rat coronal brain slices by using coupled enzymatic reactions terminating with light emission (AChE/choline oxidase/peroxidase) [56]. The developed CL reagent solution for AChE proved to be suitable for analyte localization on a target surface: it allowed direct localization of the spatial distribution of the enzyme in rat brain sections, with a sharp CL signal localized in cholinergic neurons but with very low background emission. The relative concentrations of the components of the CL reagent solution were optimized to avoid the diffusion of the light-emitting species in the surrounding solution. This imaging system could be a useful tool to study both the pathophysiological role of AChE distribution in brain and the effect of in vivo administration of enzyme-inhibiting drugs, providing a system that is more predictive than in vitro assay of inhibition activity.

5.2 Immunohistochemistry

IHC techniques provide important tools for the localization of specific antigens within individual cells. In CL IHC the probes used are highly specific antibodies that bind to antigens such as proteins, enzymes, and viral or bacterial products. The bound specific antibody is revealed indirectly by species-specific or classspecific secondary antibodies conjugated to CL enzymes.

A CL IHC technique was developed to localize interleukin 8 (IL-8) in gastric mucosa biopsy specimens, using monoclonal mouse anti-IL-8 and AP-labeled goat anti-mouse antibodies [47]. The CL IHC technique effectively localized IL- 8 in the gastric mucosa (Fig. 5b), and an increased IL-8 content was observed in epithelial cells in the presence of Helicobacter pylori infection, thus confirming earlier results achieved with an immunofluorescence technique [61]. Alternatively, the immunochemiluminescent approach could be used to identify bacterial phenotype using antisera against known strain-specific virulence determinants. Such an approach could allow identification of bacterial phenotype in situ without microbial culture. A future development, consisting of the use of double immunochemiluminescence for quantifying epithelial expression of mediators in relation to luminal pathogenic agents, has been explored.

The main advantage of using immunochemiluminescence techniques is that they allow more sensitive detection and improved localization of infectious

Figure 5 (a) Localization of endogenous AP activity in rabbit intestine cryosection by addition of CL substrate; (b) epithelial localization of interleukin 8 in gastric mucosa cryosection infected with Helicobacter pylori by immunohistochemistry; (c) localization of cytomegalovirus DNA in infected human fibroblasts by in situ hybridization. [(a) From Ref. 46. Copyright Academic Press. Reproduced with permission. (b) Courtesy of Dr. J. E. Crabtree, St. James’s University Hospital, Leeds, England.]

pathogens, and permit quantification of tissue antigens. Another advantage of the technique is that it allows examination of the immunopathogenic aspects of infections: it can be used to investigate host-pathogen interactions, in particular, inflammatory mediators in epithelial cells that are upregulated in response to infectious agents.

Immunocytochemical methods were developed to detect specific antibodies in sera from patients affected by different infectious diseases. P3-HR1 cells, which express Epstein-Barr-virus-induced virus capsid antigens (VCA), were used to search for specific human IgM (class M immunoglobulins) to VCA in infectious mononucleosis patients. After treatment of cells with serial dilutions of sera, HRP-conjugated anti-IgM antibody was added and detected with CL substrate [25].

Human embryo lung fibroblasts infected with a reference laboratory strain of herpes simplex virus (HSV) type 2 were used to detect antibody to HSV type 2 in serum samples. After treatment of cells with serial dilutions of sera, HRPlabeled immunoglobulins to human IgG (class G immunoglobulins) were added and detected with CL substrate [36]. In both cases a sharp detection of the specific antibodies was achieved with chemiluminescent assays, which proved more sensitive than the colorimetric immunoperoxidase assays.

5.3 In Situ Hybridization

ISH is a suitable method for the localization of specific nucleic acids inside individual cells with preservation of cellular and tissue morphology, thus permitting a simultaneous assessment of the morphological alterations associated with the lesion [62]. Moreover, the possibility of simultaneous detection of two or more CL probes can improve the diagnostic significance of such technique [63]. In recent years there has been a growing interest in the application of ISH for the rapid, specific, and reliable diagnosis of viral diseases, especially for those viruses that cannot be diagnosed by isolation procedures.

Different CL ISH assays for the detection of viral DNAs in tissue sections or single cells have been developed in our laboratory, taking advantage of the rapidity and sensitivity of CL detection and obtaining a reliable semiquantitative evaluation of the viral DNA presence.

A CL ISH assay for the detection of human papillomavirus (HPV) DNA was developed, in which the hybridization reaction was performed using either digoxigenin-, biotin-, or fluorescein-labeled probes [64]. The hybrids were visualized using AP as the enzyme label and a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. This assay was applied to biopsy specimens from different pathologies associated with HPV, which had previously proved positive for HPV DNA by polymerase chain reaction (PCR). The analytical sensitivity was assessed using samples of HeLa and CaSki cell lines, whose content in HPV DNA is known (10–50 copies of HPV 18 DNA in HeLa cells and 400–600 copies

of HPV 16 DNA in CaSki cells). The CL ISH assay proved sensitive and specific using either digoxigenin-, biotin-, or fluorescein-labeled probes and provided an objective evaluation of the results. CL detection was more sensitive than colorimetry, being able to detect a positive signal in HeLa cells that was negative in the colorimetric ISH assay.

B19 parvovirus DNA was detected in bone marrow cells employing digoxi- genin-labeled B19 DNA probes, antidigoxigenin Fab fragment conjugated with AP, and a 1,2-dioxetane phosphate CL substrate [32]. The CL ISH was applied to samples that had previously been tested for B19 DNA content using ISH with colorimetric detection, dot blot hybridization, and nested PCR. The CL assay proved specific and showed an increased sensitivity in detecting B19 DNA when compared to ISH with colorimetric detection, being able to find a higher number of positive cells per 100 counted cells with highly statistically significant difference.

Cytomegalovirus (CMV) DNA in cultured CMV-infected cells and in different clinical samples (tissue sections and cellular smears) was detected using digoxigenin-labeled probes, antidigoxigenin Fab fragments labeled with AP, and the adamantyl 1,2-dioxetane phenyl phosphate CL substrate [33]. The presence of hybridized CMV DNA was observed in infected cells fixed at various times after infection, and it was possible to measure increasing light emission values thus following the CMV replication cycle (Fig. 5c). When the assay was performed on clinical samples from patients with acute CMV infections, CMV DNA was detected in all the positive samples tested, in both cellular samples and tissue sections.

HSV in human fibroblasts was detected using biotin-labeled HSV DNA probes, streptavidin-HRP complex, and enhanced CL substrate reagent for HRP [56]. The presence of HSV DNA was observed in cells infected with clinical samples known to contain the HSV virus fixed at 48 h postinfection, with a sharp topographical localization and a good preservation of cellular morphology.

Chemiluminescent ISH can be used for the simultaneous detection of different DNA sequences in the same sample, by means of a double hybridization reaction in which probes to different DNA targets are labeled and detected using different enzymatic systems. The two different chemiluminescent detections have to be made sequentially, provided the first CL substrate is removed before the second CL analysis is performed by adding the other substrate. Therefore, in comparison with other existing ISH methods to detect two targets (for example, those using two different fluorescent labels), CL ISH has the important limitation of requiring sequential staining and signal visualization.

A CL ISH assay for simultaneous detection of two different viral DNAs (HSV and CMV DNAs) was developed utilizing both HRP and AP as reporter enzymes [63]. A biotinylated HSV DNA probe and a digoxigenin-labeled CMV DNA probe were cohybridized with samples; then CL detection of the two probes was performed. The HSV DNA was revealed using a streptavidin-HRP complex

amplified with biotinyl tyramide and a luminol-based CL substrate for HRP, while CMV DNA was detected by means of antidigoxigenin Fab fragments conjugated with AP and a dioxetane phosphate derivative as CL substrate. Both HSV and CMV DNAs were found in infected cells with distinct localization and absence of cross-reactions. In the double CL ISH, the enzymatic reaction of HRP is usually performed before that of AP. This is because the HRP/luminol reaction rapidly reaches a steady-state light output that is maintained for a relatively short time, while the kinetics of the AP/dioxetane reaction is slower and the CL emission lasts for a relatively long time. This detection sequence requires shorter washing to remove the HRP substrate, to avoid any interfering photon emission during the AP reaction, making the assay more rapid. Consistent results were obtained when the substrate reaction sequence was reversed; however, longer washing was required.

In conclusion, CL ISH has proved more sensitive than ISH followed by colorimetric detection and can be as sensitive as ISH using radioactive labels [31, 32, 64, 65]. When compared with fluorometric detection, CL showed an improved signal-to-noise ratio, thus a higher specific detectability; furthermore, once standardized, it provided more precise and accurate quantitative results.

6. FUTURE PERSPECTIVES

A significant improvement in the CL technique would be enhancement of the transmitted light image quality, to facilitate morphological localization of the analyte. Cryosections, generally 5–10 m thick, can be directly observed through the microscope with a phase-contrast system. A more significant improvement in image quality could also be obtained by staining the samples with appropriate dyes either after or before CL measurement, provided the staining does not interfere with the CL reaction.

Since CL imaging offers the possibility of measuring not only the light output but also its topographic distribution on a given area of the sample, it enlarges the application of biospecific reactions to simultaneous multianalyte detection by developing multiarray devices where different antigens, antibodies, or gene probes are immobilized in different areas. Miniaturized multiarray devices could be set up using chip microfabrication technology, the CL signal being evaluated by an imaging device coupled with an optical microscope or suitable optics.

An array or a matrix of nucleic acid probes immobilized at discrete locations on a silicon or glass surface provides a convenient means to simultaneously probe a sample for the presence of many different target sequences. Microarray biochip scanning devices, mostly based on fluorescent labels, are now currently available, and could also be used with CL labels to take advantage of the higher sensitivity of this detection principle.

A sensor array where different haptens are immobilized at well-defined areas on a plain glass surface has been developed [66]. Using an automated flow injection system it was possible to incubate all areas on the chip with analytes, specific antibodies, secondary HRP-labeled antibodies, and CL substrate. Measurement of the light output via imaging performed with a CCD device allowed determination of the analytes present in the sample on the basis of the spatial localization of the CL signal.

Imaging can also be useful for multiprobe detection, for example using fluorescent probes together with CL reactions. Potentially it is possible to detect first a fluorescent probe, and second detect the CL probe by adding the CL substrate. Probes marked with different CL labels, usually enzymes requiring different substrates, can also be used at the same time, provided the first CL substrate is removed before a second CL analysis is performed by adding another substrate.

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