Development of a Lateral Flow Highway: Ultra-Rapid Multitracking Immunosensor for Cardiac Markers

Abstract

The integration of several controlled parameters within a single test system is experiencing increased demand. However, multiplexed test systems typically require complex manufacturing. Here, we describe a multiplexed immunochromatographic assay that incorporates a conventional nitrocellulose membrane, which is used together with microspot printing, to construct adjacent microfluidic "tracks" for multiplexed detection. The 1 mm distance between tracks allows for the detection of up to four different analytes. The following reagents are applied in separate zones: (a) gold nanoparticle conjugates with antibodies against each analyte, (b) other antibodies against each analyte, and (c) antispecies antibodies. The immersion of the test strip in the sample initiates the lateral flow, during which reagents of different specificities move along their tracks without track erosion or reagent mixing. An essential advantage of the proposed assay is its extreme rapidity (1-1.5 min compared with 10 min for common test strips). This assay format was applied to the detection of cardiac and inflammatory markers (myoglobin, D-dimer, and C-reactive protein) in human blood, and was characterized by high reproducibility (8%-15% coefficient of variation) with stored working ranges of conventional tests. The universal character of the proposed approach will facilitate its use for various analytes.

Progress in the Optical Sensing of Cardiac Biomarkers

“…The time per assay ranged from 1.5 to 20 min [ 98 , 102 , 103 , 104 ], indicating potential for POC testing, to more than several hours ( Table 2 ). Undiluted serum was used in several LFIA [ 104 ] and homogeneous assays; however, in general, dilution with buffer was found to be an adequate procedure to bring the sample concentration in the linear range of the method and minimize interference.…”

“…At the same time, disappointingly, there was no evaluation of the stability and reproducibility of the new materials, with very few exceptions. Some lateral flow strips were evaluated and found to be stable for months at either 4 °C [ 110 ] or at room temperature [ 104 ].…”

“…In particular, major gains were related to nanomaterials’ role as catalysts (i.e., nanozymes; Refs. [ 106 , 109 ], in comparison with the more “traditional”, use as high-loading capacity carriers for colorimetric probes or recognition molecules [ 99 ] or as signaling probes themselves [ 101 , 104 ].…”

Advancements and future directions in cardiac biomarker detection using lateral flow assays

“…The reliability of the sensor was further confirmed by comparing the results of clinical samples with ELISA. Byzova et al 115 constructed a multiplex immunoassay based on multitrack on a single strip to detect D-dimer, Myo, and CRP in serum. This was achieved by adding ordered microspots for conjugate pads, test, and control zones using a steel pin pre-wetted with reagents.…”

Progress in the Optical Sensing of Cardiac Biomarkers

“…In particular, major gains were related to nanomaterials' role as catalysts (i.e., nanozymes; Refs. [106,109], in comparison with the more "traditional", use as highloading capacity carriers for colorimetric probes or recognition molecules or as signaling probes themselves [101,104].…”