Bio-chemiluminescent miniaturized biosensors

The Point-Of-Care (POC) approach is based on portable devices suitable to perform the analysis directly where it is required or where the sample is obtained. POC, which can provide significant advantages in the clinical field for its ability to provide accurate diagnosis at the patient bedside, can find applications in other fields, such as critical medicine, bio-terrorism, developing countries, food and environmental analyses, space station and telemedicine. A POC device should combine portability, minimum sample pre-treatment and ability to perform highly sensitive multiplexed assays in a short time. Microfluidic integrated systems relying on biospecific recognition reactions (e.g., immunological reactions, nucleic acids hybridization) and ultrasensitive bio-chemiluminescence (BL-CL) detection techniques represent one of the most promising options. Although several miniaturized analytical systems were developed, in most cases the miniaturization of the analytical format was not paralleled by an adequate miniaturization of the overall analytical device (e.g., use of laboratory instrumentation for signal detection). This work describes the development of POC devices exploiting “contact” BL-CL detection, in which the analytical signal is produced directly in the surface of an imaging light sensor able to localize and quantify the signal. In particular, the analytes are captured and detected in different positions of a transparent solid support, which is in contact, through a tapered fiber optic faceplate, with a portable cooled highly sensitive CCD sensor. “Contact” imaging, which provides high light collection efficiency, has been exploited in different analytical devices. A microfluidics-based device was developed for the diagnosis of parvovirus B19 (B19V) infection. In particular, target B19V DNA is captured by a specific peptide nucleic acid (PNA) probe immobilized on a glass surface, and then revealed by exploiting an enzyme label suitable for CL detection. The limit of detection (50 fmol/mL of B19V DNA) is comparable with that obtained with standard laboratory methodologies, with an overall assay time of 30 min. A new portable biodevice containing genetically engineered BL whole-cell biosensors was developed for multiplexed detection of environmental pollutants (e.g., compounds with hormone-like activity, heavy metals…). The cells were modified with the introduction of the gene encoding for a BL reporter protein (e.g., luciferase), so that its expression is controlled by the ability of the target analyte to interact with a specific regulatory proteins or receptor. The expressed reporter protein can be readily measured and directly related to the analyte concentration in the sample. Different recombinant yeast and bacterial biosensors were immobilized in a modified clear bottom black 384-well microplate to obtain a BL cell array suitable for on-site analysis. The cells array, which can be stored for up to 1 month at 4°C without loosing cell vitality, was characterized by high sensitivity (e.g., a LOD of 0.5 nM for testosterone was obtained with immobilized yeast cell-based biosensor for androgen detection after 1-month storage). The performance of “contact” imaging is evaluated and compared to that of conventional optics-based imaging performed with laboratory instrumentation.