We did found not significant changes in

We did found not significant changes in the photocurrent values, performing the experiments in different days with changes in the room temperature of about 5°C. The optimized dilution conditions for both rabbit sera and Ig-HRP were investigated. In particular, reaction buffer solutions of rabbit sera were prepared diluting the serum between 50 and 500 times and the standard solution of Ig-HRP between 200 and 2000 times. For this purpose, several microfluidic chips were functionalized with GPs, afterwards they were positioned on the top of the array of a-Si:H photosensors. The reaction buffer solutions of anti-VEA and anti-31-43 and Ig-HRP were flowed into the microchannel following the analytical procedure described in the Methods section. Finally, the ELISA assay was performed using the chemiluminescent cocktail. The results obtained from this set of experiments are summarized in Fig. 8. In this figure, the photocurrent peaks measured during the different immunoassays are reported together with the error bars, calculated over three measurements for each combination of dilution. We observed that the intensity of the photocurrent signals for the three types of interactions strongly depends on both the dilution factors. In particular, when Ig-HRP was diluted 200 times (Fig. 8a), the intensity of the CK signal for the specific and non-specific interactions is strongly different only when the rabbit antisera solution is diluted 500 times. This may depend on the possible reactions of GPs with non-specific antibody, which may occur at high concentrations of rabbit serum. Also, the intensity of the chemiluminescent signal observed when Ig-HRP is flowed into channels functionalized only with GPs (peptide-only) suggests a possible physical dna methyltransferase (non-specific absorption) of antibodies onto the PDMS and/or PHEMA polymer matrix. Similar results are obtained when Ig-HRP is diluted 2000 times (Fig. 8b). Indeed, when the rabbit antiserum is diluted 50 or 250 times, the CK signal due to non-specific interactions and absorption on channel wall is still occurring, while this undesired signal strongly decreases for rabbit antisera diluted 500 times. From Fig. 5b, we deduce that the absolute chemiluminescent peak is much lower than that achieved for Ig-HRP dilution equal to 200 and that the standard deviation is much higher. Therefore, in the investigated ranges the optimized buffer dilutions are 500 and 200 for the rabbit antisera and Ig-HRP, respectively. As a final remark, we would like to note that the total time of analysis, once the microfluidic chip has been functionalized with PHEMA-SA, is around 1h. This time includes the analytical procedure and the analysis with the array of a-Si:H photosensors, both described in the Methods section. Therefore, the presented lab-on-chip system requires half of the time that is usually necessary for an ELISA analysis [17].
Conclusions
Acknowledgments Authors thank the Center for Life Nano Scienze @ Sapienza, Istituto Italiano di Tecnologia (Rome, Italy), and the Italian Ministry of Education, University and Research (MIUR) through PRIN 2010–2011 Project ARTEMIDE (ref. 20108ZSRTR) and through University Research Project 2013 (prot. C26A13HKFB) for the financial support.
Introduction Current drinking water bacteria tests are performed in a laboratory, or use microbiological culture kits that require a minimum of 18–24h of incubation [1]. Microfluidics-based prototypes demonstrating reduced testing time have been recently reported for field monitoring of bacteria [2,3]; however, they still have relatively high detection limits (∼104cell/ml), low sample throughput and may require expensive supporting equipment. Molecular diagnostic detection methods (DNA or RNA-based) are still laboratory-based [4,5]. The ability to concentrate bacteria in-situ without culturing, or with significantly reduced culture times, is a unique competitive advantage for new technologies and key toward addressing challenges faced with bacterial culturing and detection. One way to achieve desired concentration amplification in microfluidic settings is by means of specially designed microelectrode arrays that can cause electric field effects, such as electroosmosis and dielectrophoresis [6]. Proof-of-principle demonstrations of how electric-field-assisted preconcentration strategies enable the detection of dilute target analytes are numerous [7,8]. Some characteristic examples are cited here for viruses [9,10], bacteria and yeast [11,12], DNA [13], and peptides [14].