Nanobiosensors for Monitoring Subcompartments of Single Cells

Nanosensors having antibody-based probes for use in measuring fluorescent targets inside a single cell have been demonstrated [18-23]. Since cells have very small sizes (110 /¿m), the success of intracellular investigations depends on several factors, including the sensitivity of the measurement system, the selectivity of the probe, and the small size of the nanofiber probes. The smallest cells to be nondestructively probed with a fiber-optic nanobiosensor were reported by Vo-Dinh and co-workers [21]. In that work, the antibody probe was targeted against benzopyrene tetrol (BPT), an important biological compound, which was used as a biomarker of human exposure to the carcinogen benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon of great environmental and toxicological interest because of its mutagenic/carcinogenic properties and its ubiquitous presence in the environment. Benzo[a]pyrene has been identified as a chemical carcinogen in laboratory animal studies [29]. The small size of the probe allowed manipulation of the nanosensor at specific locations within the cells. The cells were first incubated with BPT prior to measurements using the experimental procedures described previously. Interrogation of single cells for the presence of BPT was then carried out using antibody nanoprobes for excitation and a photometric system for fluorescence signal detection.

Nanobiosensors for BPT were used for the measurement of intracellular concentrations of BPT in the cytoplasm of two different cell lines: (1) human mammary carcinoma cells, and (2) rat liver epithelial cells, following treatment of the culturing media with an excess of BPT. Figure 8 shows a digital image of the nanosensor actually being inserted into a single human mammary carcinoma cell. The measurements were performed on rat liver epithelial cells (Clone 9) used as the model cell system. The cells had been previously incubated with BPT molecules prior to measurements. The results demonstrated the possibility of in-situ measurements of BPT inside a single cell.

In this study, the nanosensors employed single-use bioprobes because the probes were used to obtain only one measurement at a specific time, and could not be reused due to the strong association constant of the antibody-antigen binding process. The antibody probes, however, could be regenerated using ultrasound methods. Our laboratory has

Single Cell

Single Cell

Figure 8. Digital image of a nanosensor inserted into a single human mammary carcinoma cell (the small size of the probe allowed manipulation of the nanoprobe at specific locations within a single cell).

successfully developed a method using ultrasound to non-invasively release antigen molecules from the antibodies, and therefore to regenerate antibody-based biosensors [30]. The results of the measurements with antibody against breast cancer antigen illustrate the effectiveness and potential of the regenerable immunosensor. A 65% removal of the antigens bound to the monoclonal antibodies immobilized on the fiber surface is attained after ultrasound regeneration. The ultrasound regeneration scheme is a nondestructive approach that has a great potential to be applied to nanosensors. The results demonstrate the effectiveness of this innovative ultrasound-based approach for biosensor regeneration, that is, releasing the antigen from the antibody probe.

Multiple (e.g., five) recordings of the fluorescence signals could be taken with each measurement using a specific nanoprobe. We have made a series of calibration measurements of solutions containing different BPT concentrations in order to obtain a quantitative estimation of the amount of BPT molecules detected. For these calibration measurements, the fibers were placed in petri dishes containing solutions of BPT with concentrations ranging from 1.56 x 10-10 to 1.56 x 10-8 M. By plotting the increase in fluorescence from one concentration to the next versus the concentration of BPT, and fitting these data with an exponential function in order to simulate a saturated condition, a concentration of (9.6 ± 0.2) x 1011 M was determined for BPT in the individual cell investigated [23].

Detection of BaP transport inside single cells is of great biomedical interest since it can serve as a means for monitoring BaP exposure, which can lead to DNA damage [29]. In another study, nanosensors were developed for in-situ measurements of the carcinogen BaP [31]. In order to perform these measurements, it was necessary to use antibodies targeted to BaP. The fluorescent BaP molecules were bound by interaction with the immobilized antibody receptor, forming a receptor-ligand complex. Following laser excitation of this complex, a fluorescence response from BaP provided a basis for the quantification of BaP concentration in the cell being monitored. The fluorescence signal generated allows for a high sensitivity of detection. The intracellular measurements of BaP depend on the reaction times involved. The reaction time established in this study for antibody-BaP complexing was 5 min. This was used as a standard time to enable calibration from fiber to fiber. Additionally, the nanosensors were calibrated using standard analytical procedures using measurements of known concentration of reference solutions.

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