Microdevices integrating affinity columns and capillary electrophoresis for multibiomarker analysis in human serum

Abstract

Summary Biomarkers in human body fluids have great potential for use in screening for diseases such as cancer and diabetes, diagnosis, determining the effectiveness of treatments, and detecting recurrence. Present 96-well immunoassay technology effectively analyzes large numbers of samples; however, this approach is more expensive and less time effective on single or a few samples. In contrast, microfluidic systems are well suited for assaying small numbers of specimens in a point-of-care setting, provided suitable procedures are developed to work within peak capacity constraints when analyzing complex mixtures like human blood serum. Here, we developed integrated microdevices with an affinity column and capillary electrophoresis channels to isolate and quantitate a panel of proteins in complex matrices. To form an affinity column, a thin film of a reactive polymer was photopolymerized in a microchannel, and four antibodies were covalently immobilized to it. The retained protein amounts were consistent from chip to chip, demonstrating reproducibility. Furthermore, the signals from four fluorescently labeled proteins captured on-column were in the same range after rinsing, indicating the column has little bias toward any of the four antibodies or their antigens. These affinity columns have been integrated with capillary electrophoresis separation, enabling us to simultaneously quantify four protein biomarkers in human blood serum in the low ng/mL range using either a calibration curve or standard addition. Our systems provide a fast, integrated and automated platform for multiple biomarker quantitation in complex media such as human blood serum.

Microfluidic barcode assay for antibody-based confirmatory diagnostics

“…Put another way, nearly all antibody mass delivered to the binding sites is anticipated to be captured. For our estimated parameters (L = 100 mm, b m = 16 mM, k on = 2 6 10 5 M 21 s 21 , m i = 1 6 10 25 cm 2 V 21 s 21 ), the Da number remains larger than 10 when the E , 320 V cm 21 . With E > 320 V cm 21 the operational regime spans 10 > Da > 1, such that while the antibody delivery increases, the decline in the capture efficiency limits the total antibody capture.…”

“…Yet, microfluidic heterogeneous immunoassays specifically developed for immunoglobulin detection are limited. [20][21][22][23][24] A significant challenge stems from the high concentration levels of total immunoglobulin in serum (6-18 mg ml 21 ). [25][26][27] Underpinning the high clinical sensitivity and specificity of HCV blotting confirmatory diagnostics is the capacity to detect multiple immunoglobulin molecules in one biospecimen (i.e., multiplexing).…”

“…Microfluidic multiplexing has been accomplished in heterogeneous immunoassays via surface patterning 21,28,29 and custom functionalized beads 30 or in homogeneous immunoassays utilizing electrophoresis in microfluidic channel networks. 31 Relevant to the present study, functionalization of channel-filling polymer gels brings together advantages of homogeneous and heterogeneous assays.…”

Application of Micro-Fluidic Devices for Biomarker Analysis in Human Biological Fluids

“…Many processing steps including desalting, labeling and extraction have been successfully performed in microchip systems. The integration of an affinity column and capillary electrophoresis channels within a microdevice for the isolation and quantitation of a panel of proteins has been demonstrated by Yang et al (Yang et al, 2010) Using this microdevice, it was possible to selectively extract and analyze four proteins in spiked human blood and the system has the potential to be expanded to 30 biomarkers using additional antibodies in the affinity columns. This device is but one example of numerous new micro-fluidic devices that incorporate multiple analytical steps onto a microchip platform.…”

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

“…43,44 However, these methods require complex setups, expensive detectors, and/or complicated encoding and decoding process, 45 making them impediment for point-of-care biomarker detection for applications including disease diagnosis and therapy monitoring. Additionally, while surface modification with antibodies is crucial for achieving specificity and high sensitivity of an immunosensor, 46 it remains a large challenge in a microchannel; maintaining surface functionality and regenerating the surface modification are also difficult due to the instability of antibodies.…”