Microfluidic based immunosensor for detection and purification of carbonylated proteins

Abstract

A microchip has been developed on the basis of immno-precipitation approach for fast and sensitive enrichment of low abundant carbonylated proteins. This microfluidic method could enrich molecular biomarkers, which could be further analyzed in the proteomic study of age-related diseases and therapeutic development. In this study, an immunoaffinity-based PDMS micro-device was designed, fabricated, and chemically modified to specifically trap DNP-labeled PTM proteins of low abundance from a complex protein mixture. Carbonylated protein is selected as a representative PTM protein to illustrate the wide application of this immuno-based microchip for other PTMs which could be readily labeled by different antibody groups. Surface characterization methods such as atomic force microscopy and fluorescence microscopy were used to evaluate the construction of glutaraldehyde- and antibody- terminated PDMS substrates in the device fabrication. Quantitative study was also applied to study the target protein capture and elution efficiency of the device. In a testing mixture consisting of smaller amount of test model-In Vitro oxidized cytochrome c and large blocking protein BSA, a high sensitivity and specificity for only carbonylated protein biomarkers was demonstrated using this on-chip immnuoaffinity based extraction/enrichment. For this highly dense 193-post arrays μ-chip, a low abundance of 159 ng of standard in vitro test model- cytochrome c was enriched at flow speed of 5 μL/min within 110 min. We demonstrated that this nascent micro-immunoprecipitation (μ-IP) method is capable for enrichment of biomarkers in protein post-translation modification related diseases and promise great advance in early disease detection.

Emerging proteomic technologies for elucidating context-dependent cellular signaling events: A big challenge of tiny proportions

“…The Li group, using tryptic digests from purified alpha‐casein, has also shown that off‐chip (FeIII) IMAC micro‐columns conceptually represent an emerging and efficient enrichment platform for phosphopeptide analysis . Other nonchip methods, including microtip enrichment and serial column setups, have also been applied to metal‐affinity enrichment of phosphopeptides from as little as 1 μg of starting material . LCMD in line with microscale enrichment coupled to highly sensitive MS‐based detection may provide a path forward for qualitative and quantitative investigations of contextual cell signaling; whether the quantity of purified target proteins from microscale sample acquisition and chip‐processing workflows will be sufficiently sensitive and quantitative with current discovery LC‐MS workflows and instrumentation remains to be seen.…”

Combining Electrochemical Sensors with Miniaturized Sample Preparation for Rapid Detection in Clinical Samples

“…Examples are the polyethylene glycol modified membrane of glucose sensors that prevent components such as ascorbic acid and uric acid to reach the electrode surface and hence render the electrochemical transduction specific [ 20 , 21 ]. Also, in heterogeneous immunoassays, surfaces are blocked with polymers or proteins, such as polyvinylpyrrolidone [ 22 , 23 ], gelatin [ 22 , 24 ] casein [ 25 , 26 ], or bovine serum albumin [ 27 , 28 ], respectively. Hydrogels or similar films are often applied to not only immobilize the biorecognition element but also function as diffusion barrier for interferences from the matrix [ 29 , 30 ].…”

Fabrication and properties of silver nanowires (AgNWs) functionalized fabric

“…What's more, PDMS based microfluidic little scale devices grant cementing in a monetarily sharp manner a whole deliberate methodology diminishing its anomaly time of examination test and reagents volumes. A few vocations of PDMS-based cut back scale contraptions are accessible in the blend from the indicated lab-on-a-chip for instance devices for cells culture and examination [49] nuclear tests for the examination of nucleic acids [50,51] and proteins [52] to the alleged organ-on-a-chip [53][54][55][56]. The last referenced later application relies on microfluidic structures for the lifestyle of various cells imitating organs and complex tissues [57][58][59][60] .…”