Abstract
Paper-based colorimetric biosensors combine the use of paper with colorimetric signal detection. However, they usually demonstrate lower sensitivities because a signal amplification procedure has not been used. Stopping the reaction of colorimetric signal generation is often used in lab-based assays in order to amplify and stabilize the colorimetric signal for detection. In this study, the generation of a stopped colorimetric signal was examined for accurate and enhanced signal detection in paper-based biosensors. The colorimetric reaction in biosensors is usually based on the interaction between the enzyme horseradish peroxidase (HRP) and a selected chromogenic substrate. The two most commonly used HRP substrates, 3,3’,5,5’-tetramethylbenzidine (TMB) and 2’-azinobis (3-ethylbenzothiazoline-6-sulfonic-acid) (ABTS), were compared in terms of their ability to generate a stopped colorimetric signal on membrane. The stopped colorimetric signal was visible for TMB but not for ABTS. Moreover, the generation of stopped colorimetric signal was dependent on the presence of polyvinylidene-difluoride (PVDF) membrane as the separation layer. With PVDF the colorimetric signal (color intensity) was higher (TMB: 126 ± 6 and ABTS: 121 ± 9) in comparison to without PVDF (TMB: 110 ± 2 and ABTS: 102 ± 4). The TMB stopped colorimetric signal demonstrated a more stable signal detection with lower standard deviation values. To conclude, a stopped colorimetric signal can be generated in paper-based biosensors for enhanced and accurate signal detection.
Highlights
The three main advantages of biosensors include simplicity, cost-effectiveness and rapid results.Colorimetric detection puts to best use these important biosensors advantages
The stopped colorimetric signal was further tested with a range of horseradish peroxidase (HRP)-antibody concentrations 0–75 ng/mL (0, 0.5, 1, 2.5, 10, 25, 50 and 75 ng/mL) for with a range of HRP-antibody concentrations 0–75 ng/mL (0, 0.5, 1, 2.5, 10, 25, 50 and 75 ng/mL) for both substrates
The stopped colorimetric signal was further tested with a range of HRP-antibody concentrations from 0–75 ng/mL (0, 0.5, 1, 2.5, 10, 25, 50 and 75 ng/mL) for both substrates (Figure 3C)
Summary
The three main advantages of biosensors include simplicity, cost-effectiveness and rapid results. The colorimetric reaction in paper-based biosensors is mainly based on the interaction between the labelled antibody–protein immunocomplex and a selected chemical substrate. The secondary antibody is used in order to quantify the binding reaction between the target analyte and the specific primary antibody This specific binding interaction is detected by measuring the oxidizing reaction of HRP enzyme with a chromogenic substrate [15]. The generation of a stopped colorimetric signal was examined for an accurate and enhanced signal detection in paper-based biosensors. The stopped colorimetric signals were detected on membranes for paper-based biosensors, using a ‘Stack-Pad’ sensor layout [21,22,23], which consists of vertically stacked functional membranes (Figure 1).
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