Artificial receptors in serologic tests for the early diagnosis of dengue virus infection.

BACKGROUND: Because of the range and nonspecificity of clinical presentations of dengue virus infections, we felt there was a need to create diagnostic tests. We used artificial receptors for the virus to develop serologic assays to detect dengue virus infection. METHODS: We coated a quartz crystal microbalance (QCM) with molecularly imprinted polymers specific for nonstructural protein 1 of flavivirus. These artificial receptors were specifically created on a QCM chip by polymerization of monomers and were cross-linked in the presence of the epitope site of nonstructural protein 1. We tested serum samples from patients with confirmed cases of dengue reported to the Center for Disease Control in Taipei. Samples were diluted 100-fold; no other sample pretreatment was used. The QCM response was compared with results of monoclonal ELISA. RESULTS: QCM signals were >15 Hz in 18 of 21 (86%) of dengue samples and in 0 of 16 control samples. The correlation (r2) of the QCM response and the ELISA result was 0.73. Within-run and run-to-run imprecisions (CV) were 4%-28% and 10%-32%, respectively. CONCLUSIONS: The described assay offers a serologic technique for diagnosis of early viremia. The results illustrate the potential of well-organized polymers on the highly sensitive sensor system for diagnostic and biotechnological applications.

Recognition of dengue virus protein using epitope-mediated molecularly imprinted film.

Molecularly imprinted film was fabricated in the presence of a pentadecapeptide onto a quartz crystal microbalance (QCM) chip. This 15-mer peptide has been known as the linear epitope of the dengue virus NS1 protein. Imprinting resulted in an increased polymer affinity toward the corresponding templates but also to the virus protein. Direct detection of the dengue virus protein was achieved quantitatively. The QCM chip response to the NS1 protein was obtained using epitope-mediated imprinting demonstrating a comparable frequency shift in chips immobilized with monoclonal antibodies. The binding effect was further enhanced and confirmed using a monoclonal antibody to form a sandwich with the MIP-NS1 protein complex on the chip. No pretreatment was required.

Piezoelectric immunochip for the detection of dengue fever in viremia phase.

The global prevalence of dengue fever has grown so dramatically in recent years that it is endemic in more than 100 countries and has become a major international public health concern. Moreover, since the flu-like symptoms that accompany dengue fever are atypical and varied, the detection procedures currently used to identify it are cumbersome and time-consuming, making early stage epidemiological control and effective medical treatment of this epidemic almost impossible. In this study, a QCM-based detection system was developed in which two monoclonal antibodies against dengue E and NS-1 protein, respectively, were control orientated immobilized on QCM via protein A to produce an immunochip. Various sample pretreatment procedures were evaluated to ascertain the most suitable combination, and both the simulating samples and the clinical specimen were examined by the immunochip. The results revealed that the cibacron blue 3GA gel-heat denature (CB-HD) method was the most effective sample pretreatment technique. Due to the complex composition of the serum, the immunochip could only effectively quantify dengue viral antigens in a 1/1000 untreated simulated sample. With the help of the CB-HD method, the dilution folds were found to capable of being reduced from 1000 to 100, and the detection limit lowered to 1.727 microg/ml (E protein) and 0.740 microg/ml (NS-1 protein) in the original sample. While the cocktail immunochip could not quantify both antigens separately, the higher signal level rendered it a more effective qualification tool for suspect screening. Moreover, the results of the analysis of clinical specimens also proved the ability and future potential of cocktail immunochip in discriminating dengue-positive cases from negative serum specimens in the viremia phase.

Discrimination of peptides by using a molecularly imprinted piezoelectric biosensor.

Based on the direct formation of a molecularly imprinted polymer on gold electrodes, we have developed a peptide sensor for the detection of low-molecular-weight peptides. A new cross-linking monomer, (N-Acr-L-Cys-NHBn)(2), was employed to attach the surface of the chip and to copolymerize with other monomers. Interestingly, N-benzylacrylamide participates in the polymerization and recognition is carried out in an aqueous environment. By using quartz crystal microbalance detection, short peptides can be monitored by their interaction with plastic antibodies specific for the target peptides. The selectivity of molecularly imprinted polymers and the sensitivity of such artificial biosensors have been combined to differentiate between traces of oxytocin and vasopressin to the ng mL(-1) scale.