Extended Nanofluidic Immunochemical Reaction with Femtoliter Sample Volumes

Abstract

The growing need to optimize immunoassay performance driven by interest in analyzing individual cells has resulted in a decrease in the amount of sample required. Miniaturized immunoassays that use ultra-small femtoliter to attoliter sample volumes, a range known as the extended nanospace, can satisfy this analytical need; however, capturing every targeted molecule without loss in extended nanochannels for subsequent detection remains challenging. This is the first report of a successful extended nanofluidics-based quantitative immunochemical reaction capable of high capture efficiency using a femtoliter-scale sample volume. A novel patterning method using a photolithographic technique with vacuum ultraviolet light and low-temperature (100 °C) bonding enables patterning of functional groups for antibody immobilization before bonding, resulting in an immunochemical reaction space of only 86 fL. Reaction rate analyses indicate a decrease in the required sample volume to 810 fL and improvement in the limit of detection to 3 zmol, 5-6 orders of magnitude better than possible with the microfluidic immunoassay format. Highly efficient (near 100%) immunochemical reactions on a seconds time scale are possible due to the nm-scale diffusion length, which should be advantageous for the analysis of ultra-low-volume samples.

Materials for Microfluidic Immunoassays: A Review

“…Based on this concept, immunochemical reaction can conducts in a femtoliter volume space ( Figure 3b). [22] Glass substrate are in high demand in that the liquid in nanochannel require high pressure to drive it and the fluorescent signal require optical transparency of the substrates when fabricating an extended nanofluidic channel. However, current glass-glass bonding methods usually need high temperature and are incompatibility with functionalization (particularly those that cannot withstand high temperatures).…”

“…[43] On the other Reproduced with permission. [22] c) Digital microfluidic utilized magnetic force for separating unbound antibodies in particle-based immunoassays. Reproduced with permission.…”

Fabrication of PMMA nanofluidic electrochemical chips with integrated microelectrodes

“…The surface modification of nanochannels is another challenge, especially for the application of biosensing Shirai et al, 2014). Combining nanofluidic chips with the nanoparticle crystal (NPC) could be one of the effective approaches to the nanofluidic biosensing beacause the surface chemical properties of nanoparticles can be easily tuned.…”

Living Single Cell Analysis Platform Utilizing Microchannel, Single Cell Chamber, and Extended-nano Channel

“…[14][15][16] In addition, fL immunochemical reaction space was constructed by patterning antibodies in the extended-nano channel to realize almost 100% capture of target protein molecules. 17 The volume of the extended-nano channel (aL-fL) is much smaller than the single cell volume (pL), and the extended-nano space will be promising for living single cell analysis by analyzing small volume of sample from the single cell in the extended-nano space. However, large size gap exists between single cell and the extended-nano channel.…”