On-Chip Aptamer-Based Sandwich Assay for Thrombin Detection Employing Magnetic Beads and Quantum Dots

Abstract

In this paper, we report the development of an on-chip aptamer-based fluorescence assay for protein detection and quantification based on sandwich ELISA principles. Thrombin was selected as a model analyte to validate the assay design, which involves two DNA thrombin aptamers recognizing two different epitopes of the protein. Aptamer-functionalized magnetic beads were utilized to capture the target analyte, while a second aptamer, functionalized with quantum dots, was employed for on-chip detection. The binding of thrombin to the two aptamers via sandwich assay was monitored by fluorescence microscopy. The sandwich assay was performed on disposable microfluidic devices, fabricated on double-sided tapes and polymeric materials using a laser cutting approach. The approach enabled rapid thrombin detection with high specificity. Experimental conditions, such as reagent consumption and incubation time, were optimized in the microchip platform for the lowest limit of detection, highest specificity, and shortest assay time. The analytical performance of the microchip based assay was compared to that in the well plate format (generally utilized for ELISA-based methodologies). The results show that microfluidic chip proved to be a rapid and efficient system for aptamer-based thrombin assays, requiring only minimal (microliter) reagent use. This work demonstrated the successful application of on-chip aptamer-based sandwich assays for detection of target proteins of biomedical importance.

Immobilization of DNA on Magnetic Microparticles for Mercury Enrichment and Detection with Flow Cytometry

“…Furthermore, MMPs can be easily collected with a magnet, allowing mercury enrichment and sensor regeneration to be conveniently carried out. MMPs have been used 2 for the detection of DNA, [37,38] small molecules, [39] toxins, [40] proteins, [41][42][43] and cells. [44] For most of the assays, covalent fluorophore labeling was required for fluorescence generation.…”

Self-bending symmetric cusp beams

“…2 Nevertheless, the high nonspecific background encountered in complex media can be efficiently diminished by changing the detection format with double-site interaction, in particular, using sandwich assays. [3][4][5][6][7][8][9][10][11][12][13] Alternatively, the proximity between two aptamers that bind different thrombin epitopes has been exploited to collect a specific signal of the target. [14][15][16][17] Enhanced sensitivity and reduced reaction time have been achieved with bivalent aptamer structures in which distinct aptamers are conjugated with an optimal linker, 18,19 or co-conjugated to the sensor surface with an optimal density.…”

Biosensing of DNA oxidative damage: a model of using glucose meter for non-glucose biomarker detection

“…[1][2][3][4][5][6][7][8][9] Among them, paper-based POC devices are a special category due to the advantages of being simple, rapid, on-site, and cost-effective; they have been widely used in home health care and medical testing, [10][11][12] even environmental monitoring. [13][14][15] The lateral flow immunochromatographic assay, also called lateral flow test, is one of the simplest and most popular formats of paper-based POC devices and can be used to detect specific substances, including small molecules, [15][16][17][18] large proteins, [19][20][21] and even whole pathogens, 10 in a sample by using an immunological reaction.…”