Optical biosensing in microfluidics using nanoporous microbeads and amorphous silicon thin-film photodiodes: quantitative analysis of molecular recognition and signal transduction

Abstract

Microfluidic devices with integrated optical-to-electrical signal transduction using miniaturized photodetectors can address the need for compact and portable biosensing platforms for medical, veterinary, environmental and food safety applications. However, for different applications the features of the microfluidic point-of-need device will differ in order to be fit-for-purpose, namely the minimum detectable limits, the target sensitivity, and the detection time. Therefore, in order to perform an adequate optimization of the assay parameters for a given detection challenge, the rapid estimation of (1) equilibrium constants, (2) binding kinetics, (3) rate at which photons are generated/absorbed per captured molecule, and (4) molecular capture efficiencies is highly advantageous. This work presents a general method for the quantitative analysis of molecular capture performed on microbeads trapped inside microchannels based on a simple mass balance. Focusing on the interaction of protein A with human IgG as a model system, three different signal transduction methods, based on fluorescence, chemiluminescence and colorimetry, were compared. Acquiring the optical signal with thin-film photodiodes, the minimum detectable IgG per volume of bead solution (resin) was determined as for fluorescence, 4.4 ± 1.4 for chemiluminescence, and 34 ± 3 for colorimetry. A difference in binding efficiency for IgG-Alexa 430 and IgG-HRP was also observed by estimating the respective dissociation constants (KD) as 180 ± 40 nM and 1.6 ± 0.6 µM. Furthermore, the molecular binding (kon, koff) and enzyme kinetic parameters (kcat) were also estimated and discussed, along with considerations regarding capture yields and total assay time. Overall, this methodology proved to be simple and general to study the performance of biosensing platforms, showing potential for improving assay engineering.

Multiplexed Microfluidic Cartridge for At-Line Protein Monitoring in Mammalian Cell Culture Processes for Biopharmaceutical Production

“…The colorimetric signal is directly (sandwich) or inversely (competitive) proportional to the concentration of target protein in the sample. In this agarose bead-based setup, this colorimetric signal was previously observed to be at least 10-fold more sensitive than organic fluorophores coupled with fluorescence measurements, due to the possibility of accumulating signal over time. After developing the signal, the device is discarded since regeneration protocols to efficiently remove the captured antigens and wash the colored precipitates may hinder the performance of the biotinylated anti-IgG and anti-CHO HCP antibodies.…”

“…The continuous flow of solution minimizes mass-transport limitations along the column and reduces time to achieve equilibrium conditions. 25 , 26 After flowing the sample, a colorimetric signal is generated using a TMB-blotting substrate solution, continuously flowed through the device. The enzymatic reaction with TMB results in the formation of blue precipitates that continuously accumulate on the beads and are not washed away by the liquid flow, contrarily to soluble TMB substrate typically used in ELISAs, which would be suboptimal in this case.…”

“…At-line monitoring and quantification of key proteins, namely, CHO HCP, IgG, and LDH, throughout the cell cultivation process and subsequent downstream processing is accomplished by flowing the sample combined with detector antibodies through the beads at approximately 1 column volume per second for a certain time. The continuous flow of solution minimizes mass-transport limitations along the column and reduces time to achieve equilibrium conditions. , After flowing the sample, a colorimetric signal is generated using a TMB-blotting substrate solution, continuously flowed through the device. The enzymatic reaction with TMB results in the formation of blue precipitates that continuously accumulate on the beads and are not washed away by the liquid flow, contrarily to soluble TMB substrate typically used in ELISAs, which would be suboptimal in this case.…”

A microfluidic platform combined with bacteriophage receptor binding proteins for multiplex detection of Escherichia coli and Pseudomonas aeruginosa in blood

“…The microcolumn design comprises two channel heights to facilitate the physical entrapment of the agarose beads [72]. To obtain these devices, polydimethylsiloxane (PDMS) was employed by replicating a two-level SU-8 master, as previously described elsewhere [73]. Briefly, these include the fabrication of two aluminum masks by direct-write optical lithography (DWL) and wet etching of aluminum.…”

Regenerable Bead-Based Microfluidic Device with integrated THIN-Film Photodiodes for Real Time Monitoring of DNA Detection

“…Regeneration not only allows performing more assays on a single chip, but also enables the measurement of a control sample under the same experimental conditions. Our group has previously demonstrated that using agarose nanoporous microbeads inside microchannels enhances the sensitivity of detection of different biomarkers, mostly by increasing the available active surface area and decreasing diffusion times [3,4]. In this work, we report a regenerable microfluidic microbead-based DNA detection system with integrated fluorescence detection.…”