Early-stage Alzheimer’s disease (AD) detection from blood is still challenging due to the extremely low level of AD biomarkers in human blood because of the presence of blood-brain barrier (BBB) which restricts the direct circulation of AD molecules in the blood. The BBB is a highly selective barrie...
Early-stage Alzheimer’s disease (AD) detection from blood is still challenging due to the extremely low level of AD biomarkers in human blood because of the presence of blood-brain barrier (BBB) which restricts the direct circulation of AD molecules in the blood. The BBB is a highly selective barrier that regulates the flow of tiny passive and active fragments of AD between the brain parenchyma and the periphery blood. Consequently, the concentration of AD biomarkers in blood (i.e. amyloid beta and tau protein) are ten to hundred times lower than in CSF. Therefore, it’s is necessity to developed highly sensitivity biosensors for the diagnosis of AD in early-stage from blood for wide clinical applications. In this work, three novel electrochemical biosensors were developed and characterized for the diagnosis of AD and finally applied for clinical application detecting AD markers in blood. Firstly, a novel and low-cost wafer-scales patterned-replication micro/nanofabrication technology was developed for interdigitated electrode array (IDA) electrode on the plastic substrate. This proposed technology completely replaces traditional UV lithography for wafer-scale micro/nanofabrication. For trench-filling nanofabrication, firstly, the master mold was fabricated using conventional optical lithography with UV source and Ni-electroplating as well as a lift-off process. The hot- embossing technique was used for IDA pattern replicate to the acrylic wafer. After then a 50 nm-thin gold layer has been deposited on the pattern-replicated acrylic wafer using electron beam evaporation. Finally, adhesive tape was attached to the surface of the gold- deposited wafer, and detaching the adhesive tape had to gate a trench-filling gold IDA electrode. The fabricated IDA electrode was first demonstrated in the detection of poly- aminophenol (PAP) with 0.01 nM detection limit and then applied in the diagnosis of Alzheimer’s disease blood biomarkers; i.e., amolyed beta 42 (Ab-42) and amyloid beta aggregated (Ab. Agg) via e-ELISA using enzyme by-products PAP suitably. While quantified, the trench-filling IDA electrode was found to successfully detect Ab-42 and Ab. Agg with the lower detection limit (LOD) of 3.9 and 7.81 pg/ml. As a results, the trench-filling IDA electrode successfully diagnosis of AD but the detection limit was high. Secondly, a metal- porous graphene hybrid (MGH) nano-interdigitated array (nano-IDA) sensor was developed for the diagnosis of AD biomarkers in blood. Perhaps, this for the first time analysis of the electrochemical enzyme-linked immunosorbent assay (e-ELISA) enzyme products using MGH nano-IDA biosensor. In the e-ELISA procedure, IgG-AP was commonly utilized as an enzyme label and PAPP was an electroactive enzyme substrate for the e-ELISA. In particular, the target antigen was conjugated by the capture antibody that was coated on a 96-well microplate to make a capture antibody-target antigen complex, and then sequentially applied to the detection antibody and IgG-AP enzyme. Then upon applying the enzyme-substrate PAPP, the AP enzyme tagged at the detection antibody produced an electroactive enzyme product of PAP by reacting with the enzyme-substrate PAPP. Firstly, the developed MGH nano-IDA sensor has been demonstrated for e-ELISA in the electrochemical detection of poly- aminophenol (PAP) with 0.1 pM detection limit and then applied in the diagnosis of AD core biomarkers such as human amyloid beta-42 (Ab-42), human amyloid beta-40 (Ab- 40), human p-Tau 181 and human total tau (t-Tau). The MGH nano-IDA sensor was found to successfully detect Ab-42, Ab-40, p-Tau 181 and t-Tau with the lower detection limit (LOD) of 0.39 pg/ml, 0.78 pg/ml, 1.56 pg/ml, and 1.56 pg/ml respectively. Finally, a novel CNT-metal-porous graphene hybrid (CNT-MGH) nano- interdigitated array (n-IDA) electrochemical biosensor has been developed which successfully can detect early-stage AD markers in blood. In this case, the low-cost laser- induced graphene (LIG) technology has been used to fabricate the proposed CNT-MGH nano-IDA electrochemical biosensor sensor. In the demonstrative purpose, firstly, the CNT-MHG Nano-IDA sensor has been applied for electrochemical detection of poly- aminophenol (PAP) with a limit of detection of 0.1 pico-mole and then executed the diagnosis of AD biomarker’s p-Tau 217 and p-tau 181 in blood. The alkaline phosphatase (AP) enzyme tagged at the detection antibody produced an electroactive product of PAP by reacting with the enzyme-substrate of PAPP. Finally, the CNT-MGH nano-IDA sensor measured the current generated by the redox reaction via e-ELISA by-product PAP. While quantified, the proposed CNT-MGH nano-IDA sensor was found to successfully detect p-Tau 217 and p-Tau 181 protein in blood with the limit-of-detection (LOD) of 0.16 pg/ml and 0.08 pg/ml respectively. Therefore, it is expected that the developed MGH nano-IDA sensor might be applied to the precise diagnosis of AD markers in the blood at an early-stage.
Early-stage Alzheimer’s disease (AD) detection from blood is still challenging due to the extremely low level of AD biomarkers in human blood because of the presence of blood-brain barrier (BBB) which restricts the direct circulation of AD molecules in the blood. The BBB is a highly selective barrier that regulates the flow of tiny passive and active fragments of AD between the brain parenchyma and the periphery blood. Consequently, the concentration of AD biomarkers in blood (i.e. amyloid beta and tau protein) are ten to hundred times lower than in CSF. Therefore, it’s is necessity to developed highly sensitivity biosensors for the diagnosis of AD in early-stage from blood for wide clinical applications. In this work, three novel electrochemical biosensors were developed and characterized for the diagnosis of AD and finally applied for clinical application detecting AD markers in blood. Firstly, a novel and low-cost wafer-scales patterned-replication micro/nanofabrication technology was developed for interdigitated electrode array (IDA) electrode on the plastic substrate. This proposed technology completely replaces traditional UV lithography for wafer-scale micro/nanofabrication. For trench-filling nanofabrication, firstly, the master mold was fabricated using conventional optical lithography with UV source and Ni-electroplating as well as a lift-off process. The hot- embossing technique was used for IDA pattern replicate to the acrylic wafer. After then a 50 nm-thin gold layer has been deposited on the pattern-replicated acrylic wafer using electron beam evaporation. Finally, adhesive tape was attached to the surface of the gold- deposited wafer, and detaching the adhesive tape had to gate a trench-filling gold IDA electrode. The fabricated IDA electrode was first demonstrated in the detection of poly- aminophenol (PAP) with 0.01 nM detection limit and then applied in the diagnosis of Alzheimer’s disease blood biomarkers; i.e., amolyed beta 42 (Ab-42) and amyloid beta aggregated (Ab. Agg) via e-ELISA using enzyme by-products PAP suitably. While quantified, the trench-filling IDA electrode was found to successfully detect Ab-42 and Ab. Agg with the lower detection limit (LOD) of 3.9 and 7.81 pg/ml. As a results, the trench-filling IDA electrode successfully diagnosis of AD but the detection limit was high. Secondly, a metal- porous graphene hybrid (MGH) nano-interdigitated array (nano-IDA) sensor was developed for the diagnosis of AD biomarkers in blood. Perhaps, this for the first time analysis of the electrochemical enzyme-linked immunosorbent assay (e-ELISA) enzyme products using MGH nano-IDA biosensor. In the e-ELISA procedure, IgG-AP was commonly utilized as an enzyme label and PAPP was an electroactive enzyme substrate for the e-ELISA. In particular, the target antigen was conjugated by the capture antibody that was coated on a 96-well microplate to make a capture antibody-target antigen complex, and then sequentially applied to the detection antibody and IgG-AP enzyme. Then upon applying the enzyme-substrate PAPP, the AP enzyme tagged at the detection antibody produced an electroactive enzyme product of PAP by reacting with the enzyme-substrate PAPP. Firstly, the developed MGH nano-IDA sensor has been demonstrated for e-ELISA in the electrochemical detection of poly- aminophenol (PAP) with 0.1 pM detection limit and then applied in the diagnosis of AD core biomarkers such as human amyloid beta-42 (Ab-42), human amyloid beta-40 (Ab- 40), human p-Tau 181 and human total tau (t-Tau). The MGH nano-IDA sensor was found to successfully detect Ab-42, Ab-40, p-Tau 181 and t-Tau with the lower detection limit (LOD) of 0.39 pg/ml, 0.78 pg/ml, 1.56 pg/ml, and 1.56 pg/ml respectively. Finally, a novel CNT-metal-porous graphene hybrid (CNT-MGH) nano- interdigitated array (n-IDA) electrochemical biosensor has been developed which successfully can detect early-stage AD markers in blood. In this case, the low-cost laser- induced graphene (LIG) technology has been used to fabricate the proposed CNT-MGH nano-IDA electrochemical biosensor sensor. In the demonstrative purpose, firstly, the CNT-MHG Nano-IDA sensor has been applied for electrochemical detection of poly- aminophenol (PAP) with a limit of detection of 0.1 pico-mole and then executed the diagnosis of AD biomarker’s p-Tau 217 and p-tau 181 in blood. The alkaline phosphatase (AP) enzyme tagged at the detection antibody produced an electroactive product of PAP by reacting with the enzyme-substrate of PAPP. Finally, the CNT-MGH nano-IDA sensor measured the current generated by the redox reaction via e-ELISA by-product PAP. While quantified, the proposed CNT-MGH nano-IDA sensor was found to successfully detect p-Tau 217 and p-Tau 181 protein in blood with the limit-of-detection (LOD) of 0.16 pg/ml and 0.08 pg/ml respectively. Therefore, it is expected that the developed MGH nano-IDA sensor might be applied to the precise diagnosis of AD markers in the blood at an early-stage.
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