Constantine Anagnostopoulos

Paper-based non-mechanical valves for autonomous multi-reagent lateral flow microfluidic devices

In this paper we report the first three-dimensional (3D) and non-mechanical valve as a building block to create paper-based autonomous microfluidic devices. The valve consists of two layers of porous media that regulate flow direction. The valve also enables one fluid to gate another. By combining a single valve with 3D microchannels printed on paper, we create a paper-based autonomous device to conduct an enzyme-linked immunoassay. Furthermore, we explore new device architectures to autonomously manipulate multiple fluids by incorporating additional valves in a device.

Time Optimization of Heterogeneous Immunoassay Using PDMS Microfluidic Chip for Quantitative Detection of a Model Analyte

ABSTRACT * Corresponding author. Tel.: +1-401-874-5180; fax: +1-401-874-2355. Email address : lip@egr.uri.edu (P. Li), faghrim@egr.uri.edu (M. Faghri). Heterogeneous immunoassay (HI) has been recently used for on-chip biomolecule detection by many researchers due to its high specificity and precision. However, the protocols that have been published are too time-consuming. For HI based immunosensor to be attractive they should have short analysis times. In our work, a polydimethylsiloxane (PDMS) microfluidic device was fabricated to quantify a model analyte (bovine IgG) and used for optimizing the time required for each process. The results show that the incubation time of one single process could be reduced from typical 1 hour to 5 minutes without loss of performance. Our studies reveal that microfluidic devices are only effective in reducing incubation times for diffusion limited processes but not for interaction or adsorption limited process. 1 INTRODUCTION In the past decade, polydimethylsiloxane (PDMS) microfluidic chips have been utilized extensively in various research fields. One of the burgeoning areas is on-chip immunoassay with attractive and important applications in point-of-care diagnostics and in-the-field analysis [1]. Recent advances in immunotechnology made the detection and identification of biological analytes more convenient, more sensitive, and more specific than conventional assays [2]. The basic principle of immunoassay is the binding of antibodies to a target antigen, followed by the detection of the antigen-antibody complex. Biologists take advantage of the fact that antibodies have characteristics that make them very useful for a variety of medical and biological studies, such as the detection of food borne pathogens. In particular, antibodies typically are exquisitely specific for a particular antigen (especially well selected monoclonal antibodies) and have extremely high binding affinities for antigens. This makes them quite valuable for detecting particular antigens. Nowadays many diagnostic tests are based on immunological techniques. Of the various immunoassay formats that could possibly be used in microfluidic devices, the heterogeneous immunoassay (HI) is the predominant analytical technique for quantitative determination of a broad variety of analytes in clinical diagnosis, plant pathology, the food industry, and environment monitoring. Despite its high specificity and precision, HI is a time consuming, multi-step process that requires long incubation periods. It often takes many hours to as much as two days to perform one assay. The detection time consumed is very critical in saving people’s lives and reducing substantial economic cost on the society. Therefore, for HI based microfluidic immunosensor to be attractive they should have short analysis times. Several microfluidic chips have been developed based on HI procedures. Sato and coworkers [3] integrated an immunosorbent assay into a glass microchip for the detection of human secretory immunoglobulin A (s-IgA). They were able to reduce the time necessary for the antigen-antibody reaction from 24 h to less than 1 h. Hayes and coworkers [4] developed a HI with packed paramagnetic particles within a microchannel. The time required for direct interaction of fluorescein isothiocyanate (FITC) with immobilized anti-FITC was only 3 min. Lai and coworkers [5] reported a compact disk-like microfluidic platform that performed HI for rat IgG from a hybridoma cell culture. The enzymatic reaction took only 200 seconds (120 min in a microtiter plate). The reason for these dramatic reductions in processing time is because the diffusion time required for the molecule to adsorb onto the solid surface is proportional to the square of the characteristic length of the channel [6]. In the published literature, only the specific antigen-antibody reaction time has been optimized among all HI steps. However, the time required for other steps such as coating, blocking, and washing should also be optimized as the

Fluorescence Amplification Using Biospecific Nanoparticle Conjugates as Labels in Microfluidic Heterogeneous Immunoassays

In microfluidic sensing systems, it is challenging to achieve desirable sensitivity for detecting reduced number of analytes in a small volume (pL–nL). Many efforts have been made in order to improve the sensitivity of microfluidic fluorescence detection systems. Some prevalent methods (e.g., optical components integration and analyte enrichment) either increase cost or require extra operational steps. Fluorescence amplification using dye-doped silica nanoparticles has proved to be an inexpensive and efficient approach; however, this technique is still far from perfect. For instance, dye molecules physically entrapped in the silica nanoparticles occasionally leak and can cause false-negative results. In addition, the nonspecific adsorption of nanoparticles has not been resolved.Copyright

A Blocking-Free Microfluidic Fluorescence Heterogeneous Immunoassay for Quantitative Detection of Human C-Reactive Protein

In this article, a rapid, sensitive, and disposable microfluidic immunosensor is presented for point-of-care testing and clinical diagnostics. For the first time, a heterogeneous immunoassay without blocking process is achieved by using protein A functionalized polydimethylsiloxane microchannels. C-reactive protein (CRP), a biomarker for inflammation and cardiovascular disease risk assessment, is selected as a model analyte to demonstrate the sensitivity of this blocking-free microfluidic heterogeneous immunoassay. A four parameter logistic function is used to model and assess the data. The limit of detection obtained is 0.36 μg/mL, which is lower than the cut-off value for clinical diagnosis. The overall assay is completed in 23 min. The assay procedure can be modified for detection of other disease biomarkers or virulent pathogens by using different capture and detection antibodies.Copyright

HETEROGENEOUS DETECTION OF PCR-AMPLIFIED INTIMIN GENE FROM E. COLI O157:H7 VIA PDMS MICROFLUIDIC CHIP

E. coli O157:H7 strains represent the most important group of food-borne pathogens. PCR-amplified intimin gene of pathogenic E. coli O157:H7 was detected heterogeneously via a microfluidic chip that consists of streptavidin-coated nanoliter chambers. Biotinylated primers and digoxigenin labeled deoxyuridine triphosphate (dUTP) were incorporated into the amplified intimin (eaeA) gene by an off-chip PCR thermal cycler. The amplified products were injected into the chip where they were immobilized via streptavidin-biotin interaction. Detection of the products using alkaline phosphatase (AP) conjugated anti-digoxigenin was performed with an epi-fluorescent microscope. This assay was capable of detecting 0.06 ng/μL biotin-digoxigenin-dsDNA conjugate distinctly, which is a hundred fold more sensitive than the traditional detection by agarose gel.Copyright