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A highly sensitive plasma-based amyloid-β detection system through medium-changing and noise cancellation system for early diagnosis of the Alzheimer’s disease

We developed an interdigitated microelectrode (IME) sensor system for blood-based Alzheimer’s disease (AD) diagnosis based on impedimetric detection of amyloid-β (Aβ) protein, which is a representative candidate biomarker for AD. The IME sensing device was fabricated using a surface micromachining process. For highly sensitive detection of several tens to hundreds of picogram/mL of Aβ in blood, medium change from plasma to PBS buffer was utilized with signal cancellation and amplification processing (SCAP) system. The system demonstrated approximately 100-folds higher sensitivity according to the concentrations. A robust antibody-immobilization process was used for stability during medium change. Selectivity of the reaction due to the affinity of Aβ to the antibody and the sensitivity according to the concentration of Aβ were also demonstrated. Considering these basic characteristics of the IME sensor system, the medium change was optimized in relation to the absolute value of impedance change and differentiated impedance changes for real plasma based Aβ detection. Finally, the detection of Aβ levels in transgenic and wild-type mouse plasma samples was accomplished with the designed sensor system and the medium-changing method. The results confirmed the potential of this system to discriminate between patients and healthy controls, which would enable blood-based AD diagnosis.

2 Fig. S1. Equivalent circuit of IME sensor and cancellation principle.
The designed interdigitated microelectrode was used for Aβ protein detection with signal processing systems. The interaction between Aβ protein and Aβ antibody, which is immobilized at the functional electrode of the sensor, led to changes in the impedance. Fig. S1.
(a) shows an equivalent circuit of IME sensor in buffer solution before the functional electrode for accurate measurement of impedance changes by the interaction between Aβ protein and Aβ antibody (Z biomolecule ). The biomolecule-containing liquid has high impedance. The designed ∑ + -3 system was utilized to cancel out the impedance of liquid (Z liquid ) and the specified parasitic impedance caused by the system and device. The Z system and Z contact, which respectively represent impedance due to the measurement system and measuring probe and electrode, were considered in the equivalent circuit. The Z device represents the impedance between electrodes in a single device. As shown in Fig. S1, the reference electrode was used to cancel out these all impedances for enhancement of sensitivity. As shown in Fig. S1(b), the voltages from the functional electrode (V functional ) and reference electrode (V reference ) were calculated to cancel out other impedances for the accurate measurement of impedance changes by only the Z biomolecule with the system. The output voltage (V out ), which is due to the Z biomolecule , was also amplified with the amplifier after the cancellation between the voltages of functional and reference electrodes as differential amplifier works. After obtaining an Aβ antibody layer on SiO 2 , the interaction between the immobilized Aβ antibody and Aβ antigen could be measured using the designed IME sensing system. The designed sensing system, which has a signal cancellation and amplification process (SCAP) system embedded in the measurement system, was accomplished as shown in Fig. S2(a) in order to enhance sensitivity along with noise removal. Generally, the noise occurred by a biomolecule-containing solution and the structure of IME, which has a relatively long electrode length between electrodes. The Z liquid and Z biomolecule marked in Fig. S2 respectively. The Z liquid and Z biomolecule were more dominant than other impedance components, as shown in an equivalent circuit (see Fig. S1(a) for detatils.). The effect of Z liquid was approximately 1000-2000 fold greater than that of Z biomolecule . Therefore, we developed a cancellation and amplification process to remove Z liquid and other impedance components.
Functional and reference electrodes were utilized. The functional electrode could detect Z biomolecule , including the effect of liquid, Z liquid . The reference electrode only detected Z liquid . Therefore, we loaded the biomolecule-containing solution at the functional electrode and the same liquid without biomolecules at the reference electrode. The same conditions, voltage, and frequency were applied at both electrodes. After measurement of two impedimetric signals by the functional and reference electrodes, the two signals (Z biomolecule/ Z liquid and Z liquid ) were subtracted, to obtain the changes that were due to biomolecules (Z biomolecule ). The amplification was also applied after the cancellation. Finally, we acquired amplified signals obtained only due to biomolecules (Z biomolecule ) with the designed measurement systems. Other modes that do not apply cancellation and amplification could also be used.
To verify IME impedance measuring system, we repeatedly compared SCAP system with commercial equipment (PGSTAT302N, Metrohm Autolab), as shown in Fig. S2(b). After 10 pg mL-1 Aβ-Aβ antibody interaction, the impedance change of the same IME was monitored using each measurement system. About 2.6%, 2.7%, and 4.3% of impedance changes were measured using commercial equipment, IME without SCAP system, and IME with SCAP system, respectively; approximately 1.9%, 2%, and 1% standard deviations, respectively, were calculated for the impedance changes. By utilizing IME with SCAP system, we achieved a 1.6-fold higher impedance change and a half lower standard deviation compared with other systems. We also confirmed that the IME with SCAP system for Aβ detection was more suitable than the other systems.  Before the application for IME sensor, the impedance measuring system with SCAP is evaluated. First, we prepared and compounded the electrical components (resistor of 0.1, 0.5, 1, 5, 10, 50, 100, 200 MΩ and capacitor of 10, 50, 100, 500 pF). The conductance and capacitance were monitored with SCAP and commercial equipment (Agilent 4980) as shown Fig. S3. When the conductance and the capacitance measured, the ratio of error of conductance and capacitance with SCAP is reduced as described in Table. Furthermore, IME's resistance of approximately 0.4 MΩ and capacitance of 170 pF were measured, respectively. In the capacitance of range 100 to 500 pF and resistance of range 0.1 to 0.5 MΩ, the impedance measuring system with SCAP is more appropriate with low measuring error.