Development of a novel microfluidic immunoassay for the detection of Helicobacter pylori infection

Abstract

The miniaturization of laboratory processes offers substantial advantages over traditional techniques in terms of cost, speed, and potential for multistage automation. To date, only a few studies have reported successful microfluidics-based immunoassays, most of which rely on fluorescence detection technologies. The goal of this study was to develop a poly(dimethylsiloxane) microfluidics-based immunoassay methodology and a versatile colorimetric quantification scheme for the detection of visual colour changes resulting from immune reactions in microchannels. The novel immunoassay technique was applied towards the detection of Helicobacter pylori infection using 20 human serum samples of known infection status, and results compared with conventional nitrocellulose membrane-based dot-ELISA. The microchannel immunoassay reliably detected H. pylori antigens in quantities on the order of 10 ng, which provides a sensitivity of detection comparable to conventional dot-blot assays. Sensitivity was 100%, specificity was 90%, positive predictive value 91%, and negative predictive value 100%, with an overall accuracy of 95%. The software developed generated results that were consistent with visual observations and by automatically taking into account background intensity changes, the software minimized subjectivity. Volumes of solutions used were 100-fold less compared with conventional immunoassays. Miniaturization of the ELISA using this technique provides a means for the accurate diagnosis of microbial infection while minimizing waste production.

Biomarkers and diagnostic tools for detection of Helicobacter pylori

“…However, when these serological kits were tested in Asian or middle east countries, the diagnostic accuracy of these tests were found to be lower, such as reports from Chinese (Leung et al 1999) and Japanese patient populations (Miwa et al 2001). These differences may be due to some reasons, such as crossreactivity to other intestinal bacteria which vary in Lin et al (2004), andPereira et al (2010) different parts of the world, the presence of H. pylori strain heterogeneity in different geographic areas (Ito et al 1997), and varying immunological responses to antigenic proteins of H. pylori in different patient populations (Khanna et al 1998). A lot of money is being spent on purchasing diagnostic kits which may not be suitable for patients in other countries and may not be as sensitive as kits made from antigens using locally isolated H. pylori Meijer et al 1997b).…”

“…4b). The assay time was only 25 min (Gao et al 2005;Kakaç et al 2010;Lin et al 2004). Lin et al (2004) also employed a pressure-driven microfluidic platform for rapid detection of H. pylori.…”

“…The assay time was only 25 min (Gao et al 2005;Kakaç et al 2010;Lin et al 2004). Lin et al (2004) also employed a pressure-driven microfluidic platform for rapid detection of H. pylori. The system could provide accurate readings within 30 min, while the required solution volumes were 100-fold less than conventional ELISA systems (Fig.…”

Development of a Nanoparticle-Labeled Microfluidic Immunoassay for Detection of Pathogenic Microorganisms

“…The procedures used for bacterial culture and antigen preparation were described previously (13,14). Briefly, H. pylori strain ATCC 49503 was cultured on Columbia agar plates containing 5% sheep blood, and the plates were incubated at 37°C under microaerophilic conditions (5% oxygen, 85% nitrogen, and 10% carbon dioxide) for 72 h (13) Bacteria were then inoculated into brucella broth supplemented with 10% fetal bovine serum and were grown overnight with gentle shaking under microaerophilic conditions at 37°C.…”

Engineering of PDMS surfaces for use in microsystems for capture and isolation of complex and biomedically important proteins: Epidermal growth factor receptor as a model system

“…These studies revealed that >95% of the captured mouse IgG was transferred to the glass. While specific capture of several other antigens to antibodies immobilized on PDMS has been demonstrated qualitatively, 15 quantitative data of the type described above for mouse IgG is lacking in the literature dealing with complex proteins such as membrane receptor proteins. In this paper, we provide such data utilizing a biomedically relevant protein associated with the development of human cancers, namely the epidermal growth factor (EGF) receptor.…”