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Harpaz, D. - School of Material Science and Engineering, Nanyang Technology University, 50 Nanyang Avenue, Singapore, 639798, Singapore, Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel, Institute for Sports Research (ISR), Nanyang Technology University and Loughborough University, Nanyang Avenue, Singapore, 639798, Singapore; 
Ng, T.S.E. - School of Material Science and Engineering, Nanyang Technology University, 50 Nanyang Avenue, Singapore, 639798, Singapore;
Marks, R.S. - Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel, National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel, Ilse Katz Centre for Meso and Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel;
Tok, A.I.Y. - School of Material Science and Engineering, Nanyang Technology University, 50 Nanyang Avenue, Singapore, 639798, Singapore, Institute for Sports Research (ISR), Nanyang Technology University and Loughborough University, Nanyang Avenue, Singapore, 639798, Singapore

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.

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Enhanced colorimetric signal for accurate signal detection in paper-based biosensors
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Harpaz, D. - School of Material Science and Engineering, Nanyang Technology University, 50 Nanyang Avenue, Singapore, 639798, Singapore, Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel, Institute for Sports Research (ISR), Nanyang Technology University and Loughborough University, Nanyang Avenue, Singapore, 639798, Singapore; 
Ng, T.S.E. - School of Material Science and Engineering, Nanyang Technology University, 50 Nanyang Avenue, Singapore, 639798, Singapore;
Marks, R.S. - Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel, National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel, Ilse Katz Centre for Meso and Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel;
Tok, A.I.Y. - School of Material Science and Engineering, Nanyang Technology University, 50 Nanyang Avenue, Singapore, 639798, Singapore, Institute for Sports Research (ISR), Nanyang Technology University and Loughborough University, Nanyang Avenue, Singapore, 639798, Singapore

Enhanced colorimetric signal for accurate signal detection in paper-based biosensors

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.

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