Lateral Flow Aptasensor for Simultaneous Detection of Platelet-Derived Growth Factor-BB (PDGF-BB) and Thrombin

Abstract

Here we report a lateral flow aptasensor (LFA) for the simultaneous detection of platelet-derived growth factor-BB (PDGF-BB) and thrombin. Two pairs of aptamers, which are specific against PDGF-BB and thrombin, respectively, were used to prepare the LFA. Thiolated aptamers were immobilized on a gold nanoparticle (AuNP) surface and biotinylated aptamers were immobilized on the test zones of an LFA nitrocellulose membrane. The assay involved the capture of PDGF-BB and thrombin simultaneously in sandwich-type formats between the capture aptamers on the test zones of LFA and AuNP-labeled detection aptamers. AuNPs were thus captured on the test zones of the LFA and gave red bands to enable the visual detection of target proteins. Quantitative results were obtained by reading the test band intensities with a portable strip reader. By combining the highly specific molecular recognition properties of aptamers with the unique properties of lateral flow assay (low-cost, short assay time and a user-friendly format), the optimized aptasensor was capable of simultaneously detecting 1.0 nM of PDGF-BB and 1.5 nM of thrombin in association with a 10-min assay time. The biosensor was also successfully applied to detect PDGF-BB and thrombin in spiked human serum samples. The LFA shows great promise for the development of aptamer-based lateral flow strip biosensors for point-of-care or for the in-field detection of disease-related protein biomarkers.

The Challenges of Developing Biosensors for Clinical Assessment: A Review

“…The illustration of the specificity performance in biosensors can be depicted in Figure 9. Several attempts have been reported to improve the specificity and low-fouling binding during the biosensing measurements, either using a competitive assay or complex media such as serum or whole blood, as well as blocking assays [88][89][90], a polymer brush, and a zwitterionic functionalization surface [91][92][93][94], or by using label and sandwich assays to obtain a specific signal [95][96][97]. In immunoassays, monoclonal antibodies are also recommended to obtain high specificity and lower cross-reactivity events [98].…”

The challenges of developing biosensors for clinical assessment: a review

“…For example, by blocking assays [88][89][90], polymer brush, and zwitterionic functionalization surface [91][92][93][94], also by using label and sandwich assays to obtain a specific signal [95][96][97]. In immunoassays, monoclonal antibodies are also recommended to obtain high specificity and lower cross-reactivity events [98].…”

Label-Free Quartz Crystal Microbalance Biosensor Based on Aptamer-Capped Gold Nanocages Loaded with Polyamidoamine for Thrombin Detection

“…5,6 Moreover, recent studies have made progress in thrombin detection, including enzyme-linked sandwich assays, 7,8 strips, 9−11 optical sensors, 12−14 electrochemical platforms, 15−17 and piezoelectric methods. 18 In general, enzyme-linked sandwich assays are the gold standard for thrombin detection 11 but also experience laborious steps, low analysis speed, and false positives. Lateral flow assays (strips) are rapid, cost effective, and great for point-of-care (POC) tests but usually have higher detection limits or qualitative results without additional professional equipment.…”

“…Thrombin is a serine protease, an enzyme that plays a key role in blood coagulation, and is relevant to a plethora of diseases such as chronic inflammatory diseases, vascular disease, coagulation abnormalities, cancer, and atherosclerosis. − Generally, excessive thrombin will cause venous thrombosis, and a low amount of thrombin will increase bleeding risks . The concentration of thrombin during coagulation varies from picomolar to micromolar levels, and thus, the limit of detection (LOD) and detection range need to cover the picomolar to micromolar range. , Moreover, recent studies have made progress in thrombin detection, including enzyme-linked sandwich assays, , strips, − optical sensors, − electrochemical platforms, − and piezoelectric methods . In general, enzyme-linked sandwich assays are the gold standard for thrombin detection but also experience laborious steps, low analysis speed, and false positives.…”

“…The concentration of thrombin during coagulation varies from picomolar to micromolar levels, and thus, the limit of detection (LOD) and detection range need to cover the picomolar to micromolar range. , Moreover, recent studies have made progress in thrombin detection, including enzyme-linked sandwich assays, , strips, − optical sensors, − electrochemical platforms, − and piezoelectric methods . In general, enzyme-linked sandwich assays are the gold standard for thrombin detection but also experience laborious steps, low analysis speed, and false positives. Lateral flow assays (strips) are rapid, cost effective, and great for point-of-care (POC) tests but usually have higher detection limits or qualitative results without additional professional equipment. , Electrochemical and piezoelectric platforms are highly sensitive and could achieve real-time monitoring but often require multiple separation and washing steps for detection .…”