A multiplexed immunoaggregation biomarker assay using a two-stage micro resistive pulse sensor

Abstract

We present an immunoaggregation assay chip for multiplexed biomarkers detection. This chip is based on immunoaggregation of antibody functionalized microparticles (Ab-MPs) to quantify concentrations of multiple biomarkers simultaneously. A mixture of multiple types of Ab-MPs probes with different sizes and magnetic properties, which were functionalized by different antibodies, was used for the multiplexed assay. The interactions between biomarkers and their specific Ab-MPs probes caused the immunoaggregation of Ab-MPs. A two-stage micro resistive pulse sensor was used to differentiate and count the Ab-MP aggregates triggered by different biomarkers via size and magnetic property for multiplexed detection. The volume fraction of each type of Ab-MP aggregates indicates the concentration of the corresponding target biomarker. In our study, we demonstrated multiplexed detection of two model biomarkers (human ferritin and mouse anti-rabbit IgG) in 10% fetal bovine serum, using anti-ferritin Ab and anti-mouse IgG Ab functionalized MPs. We found that the volume fraction of Ab-MP aggregates increased with the increased biomarker concentrations. The detection ranges from 5.2 ng/ml to 208 ng/ml and 3.1 ng/ml to 5.12 Â 10 4 ng/ml were achieved for human ferritin and mouse anti-rabbit IgG. This bioassay chip is able to quantitatively detect multiple biomarkers in a single test without fluorescence or enzymatic labeling process and hence is promising to serve as a useful tool for rapid detection of multiple biomarkers in biomedical research and clinical applications. V C 2016 AIP Publishing LLC. [http://dx

A microfluidic competitive immuno-aggregation assay for high sensitivity cell secretome detection

“…Therefore, the competitive immuno-aggregation method presented in this paper has three major advantages: 1) this method has a lower detection limit (0.01 ng/mL) than the method in our previous work. 57,58 This is because our previous study used non-competitive molecular binding between recognition biomolecules and target proteins. Non-competitive binding usually requires a large amount of target proteins to initiate the formation of MP aggregates, which could compromise the sensitivity of the assay.…”

“…The equilibrium dissociation constant of the binding between VEGF and VEGF Ab was reported to be 9.94 pM 56 The commercial VEGF Abs have comparable binding affinities; 2) the high binding capacity of the MPs because of their large surface-to-volume ratio. Additionally, in contrast to the non-competing aggregation method, 57,58 this assay generated a monotonic standard curve without any saturation effect; i.e., when the target protein saturated and blocked the surface of the MPs, fewer aggregates can be formed with the blocked microparticles. 57,58 Thus, for the competitive immuno-aggregation method, the corresponding VEGF concentration and the average volume change of aggregates/particles forms an injective relation.…”

“…Additionally, in contrast to the non-competing aggregation method, 57,58 this assay generated a monotonic standard curve without any saturation effect; i.e., when the target protein saturated and blocked the surface of the MPs, fewer aggregates can be formed with the blocked microparticles. 57,58 Thus, for the competitive immuno-aggregation method, the corresponding VEGF concentration and the average volume change of aggregates/particles forms an injective relation. The detection limit of this method (0.01 ng/mL) was also lower than that (0.1 ng/mL) of the noncompeting aggregation method.…”

Lab-on-a-chip electrical multiplexing techniques for cellular and molecular biomarker detection

“…Recently microparticle based immuno-aggregation methods [107][108][109][110][111][112] have been developed to (1) amplify the size change caused by specific binding and (2) enable multiplexed detections of multiple biomarkers. Han et al have used microparticles with different sizes and magnetic property as platforms for immuno-aggregation.…”

“…Han et al have used microparticles with different sizes and magnetic property as platforms for immuno-aggregation. 112 The method can quantify concentrations of multiple biomarkers simultaneously based on measuring the immuno-aggregates of antibody functionalized microparticles (Ab-MPs). Further Han et al developed a two-stage micro Coulter counter [as shown in Fig.…”