A single snapshot multiplex immunoassay platform utilizing dense test lines based on engineered beads

Abstract

Co-circulation of coronavirus disease 2019 (COVID-19) and dengue fever has been reported. Accurate and timely multiplex diagnosis is required to prevent future pandemics. Here, we developed an innovative microfluidic chip that enables a snapshot multiplex immunoassay for timely on-site response and offers unprecedented multiplexing capability with an operating procedure similar to that of lateral flow assays. An open microchannel assembly of individually engineered microbeads was developed to construct nine high-density test lines, which can be imaged in a 1 mm 2 field-of-view. Thus, simultaneous detection of multiple antibodies would be achievable in a single high-resolution snapshot. Next, we developed a novel pixel intensity-based imaging process to distinguish effective and non-specific fluorescence signals, thereby improving the reliability of this fluorescence-based immunoassay. Finally, the chip specifically identified and classified random combinations of arbovirus (Zika, dengue, and chikungunya viruses) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies within 30 minutes. Therefore, we believe that this snapshot multiplex immunoassay chip is a powerful diagnostic tool to control current and future pandemics.

Microfluidic systems for the analysis of blood‐derived molecular biomarkers

“…The versatility and advantages offered by nano/microbeads integrated in microfluidic devices have produced highly sensitive and multiplexed assays, some of them as POC platforms. For example, a device based on microbeads called MOnITOR can simultaneously detect antibodies against arboviruses (zika virus, dengue, and chikungunya) and SARS-CoV-2 in 30 min [253]. Another example is the volumetric bar-chart microfluidic chips (V-Chips), which display results in the form of bar graphs and are easy to visualize without the need for external optical instruments [248,259].…”

“…Commonly, microbeads are used in suspension because their size allows one to mix them appropriately with the assay reagents and then be easily identified with flow cytometry [213,249,252]. When used in microfluidics, beads are typically retained inside the device by either (i) physical barriers patterned within the microfluidic chan-nel [253] and (ii) biorecognition approaches [214,231], or (iii) by external magnetic fields while using paramagnetic beads [215,254,255]. Additionally, nano/microbeads are chemically stable and easy to mass-produce using PMMA, gold, or polystyrene materials.…”

“…Another strategy to improve the LOD of microfluidicbased immunoassays is to increase the area available to immobilize biorecognition elements. In microfluidics, patterning nano/microstructures, such as bead or particle packing [251,253], nanowires [240,277], and carbon nanotubes [278,279], can greatly increase the available area for the immobilization of biorecognition elements. This approach has been shown to increase the number of immobilized molecules 100-fold compared to flat surfaces [209,214,215].…”

Development of SLAF-Sequence and Multiplex SNaPshot Panels for Population Genetic Diversity Analysis and Construction of DNA Fingerprints for Sugarcane

“…SNaPshot is an easily integrable SNP typing option for forensic laboratories that has readily available forensic human and non-human DNA assays [ 25 ]. At present, SNaPshot has been widely used in bacterial community identification [ 26 ], human disease correlation [ 27 ], and forensic medicine, but to date has not been widely used for plant genetics. In Arabidopsis thaliana , 17 uniformly-distributed SNP markers were constructed using multiple SNaPshot techniques were used to identify different varieties [ 28 ].…”

Conformal Hydrogel-Skin Coating on a Microfluidic Channel through Microstamping Transfer of the Masking Layer

“…5,6 Microfluidic architectures have mostly been constructed using elastomers, such as polydimethylsiloxane (PDMS), which has good biocompatibility, transparency, formability, and straightforward fabrication-related attributes, including curing and peel-off. 2,7 PDMS has a hydrophobic surface that has been utilized for oil−water separation and water-in-oil emulsification. 8,9 However, limitations of the intrinsic hydrophobic surface, including nonspecific fouling 10 and the need for bulky off-chip equipment for flow generation, 11 inhibit the progress in advanced applications that require hydrophilic surfaces, such as oil-in-water emulsification 12 and point-of-care (POC) diagnostics.…”