Earlier Detection of Alzheimer’s Disease Based on a Novel Biomarker cis P-tau by a Label-Free Electrochemical Immunosensor

Early detection of cis phosphorylated tau (cis P-tau) may help as an effective treatment to control the progression of Alzheimer’s disease (AD). Recently, we introduced for the first time a monoclonal antibody (mAb) with high affinity against cis P-tau. In this study, the cis P-tau mAb was utilized to develop a label-free immunosensor. The antibody was immobilized onto a gold electrode and the electrochemical responses to the analyte were acquired by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and differential pulse voltammetry (DPV). The immunosensor was capable of selective detection of cis P-tau among non-specific targets like trans P-tau and major plasma proteins. A wide concentration range (10 × 10−14 M–3.0 × 10−9 M) of cis P-tau was measured in PBS and human serum matrices with a limit of detection of 0.02 and 0.05 pM, respectively. Clinical applicability of the immunosensor was suggested by its long-term storage stability and successful detection of cis P-tau in real samples of cerebrospinal fluid (CSF) and blood serum collected from human patients at different stages of AD. These results suggest that this simple immunosensor may find great application in clinical settings for early detection of AD which is an unmet urgent need in today’s healthcare services.


Introduction
Alzheimer's disease (AD) is a chronic, devastating dysfunction of neurons in the brain characterized by progressive memory impairment, noticeable personality changes, cognitive impairment, dementia, and eventual death [1]. AD represents one of the biggest medical challenges over the past five decades [1][2][3][4]. According to the World Health Organization (WHO), more than 50 million people suffer from dementia and this number is expected to exceed 152 million by the year 2050 [5,6]. One of the biggest challenges in control of AD is the early diagnosis of disease which is crucial for effectiveness of the drugs and subsequently less nerve damage [4,[7][8][9][10]. By early detection of AD in the first stages of the disease, the routine treatments can be more effective to prevent disease progression and reduce the mortality and healthcare costs [9,10]. electrochemical analyses. This label-free detection mechanism allows for easy, fast, and cost-effective analysis without the need for labeling the analyte with the signal tags (e.g., enzymes and metal nanoparticles).
Our result shows applicability of this simple biosensor in clinical settings for early detection of AD.

Electrode Preparation
For the purpose of removing and cleaning any impure materials from the GDE surface, first, the bare electrodes were polished with alumina powders (5.0, 0.30, and 0.1 µm) for 5 min with each of the sizes, subsequently immersed in piranha solution (1:3, 30% H 2 O 2 and concentrated 98% H 2 SO 4 ) for 5 min, and then rinsed with deionized water. To achieve a maximum cleaning, the GDE was cleaned by electrochemical method in 0.5 M NaOH solution via cyclic voltammetry (CV) at 0.1 V/s scan rate and in the −0.3 to 0.8 V potential range. Then electrodes were immersed into 0.5 M H 2 SO 4 and CV conducted at 0.1 V/s scan rate and in the −0.3 to 0.8 V potential range. After the processes, the GDE was washed with deionized water and dried.

Fabrication of Immunosensor
Cis P-tau mAb, the same clone as #113 from Creativebiolabs, was produced with 99% purity in the lab according to the procedure of our previous publications [57]. The immunosensor was fabricated by covalent immobilization of the antibody on the surface of the gold electrode ( Figure 1). Two thioacid cross-linkers (MPA and MUA) were used to establish attachment sites on the gold for antibody immobilization. A mixture of MUA/MPA (70/30) in ethanol (10 mM) was dropped on the gold surface and dried for 8 h at room temperature (RT). The linker-modified electrode was washed by ethanol to remove all non-bonded molecules. Then, a mixture of EDC (20 mM)/NHS (5 mM) in 0.1 M PBS (pH = 5.5-6) was dropped onto the surface at RT and left to dry. Next, a PBS solution of cis P-tau mAb (10 µg/mL, pH 7.5) was dropped onto surface and kept overnight at 4 • C to dry and then rinsed with PBS solution. In the last stage, to block non-specific adsorption, a PBS solution of BSA (1% w/v) was dropped onto the electrode surface, kept for1 h, and rinsed with PBS. The immunosensors based on SPGE were fabricated via a similar protocol, without need for the initial electrode cleaning. The fabricated immunosensors were stored at 4 • C until use. tion of cis P-tau mAb (10 µg/mL, pH 7.5) was dropped onto surface and kept overnight at 4 °C to dry and then rinsed with PBS solution. In the last stage, to block non-specific adsorption, a PBS solution of BSA (1% w/v) was dropped onto the electrode surface, kept for1 h, and rinsed with PBS. The immunosensors based on SPGE were fabricated via a similar protocol, without need for the initial electrode cleaning. The fabricated immunosensors were stored at 4 °C until use.

Morphological Characterization of Immunosensor Surface
In each step of the immunosensor assembly, the surface morphologies of the electrodes were analyzed over a 10 × 10 µm area by atomic force microscopy (AFM; VEECO CP II, Veeco Instruments Inc., New York, NY, USA). The imaging process was operated in contact mode, and scanned at a rate of 1.0 Hz. To show the capability for cis P-tau interaction with the immobilized antibody, the modified electrode was subjected to incubation with the cis P-tau solution in PBS (1.0 × 10 −12 M) for 30 min at RT, followed by washing with 5 mL of PBS before imaging.

Electrochemical Characterization of Immunosensor
Electrochemical impedance spectroscopy (EIS) and CV were used to monitor the electrochemical performances of immunosensor at different steps of the layer-by-layer procedure of immunosensor fabrication. The electrochemical measurements were conducted in 10 mM K4Fe(CN)6/K3Fe(CN)6 (1:1 ratio) by using a three-electrode set up including the modified Au electrode (1.5 mm 2 surface area) as working electrode (WE), a Pt wire as an auxiliary electrode (AE), and Ag/ AgCl as the reference electrode (RE). The EIS and CV were carried out using an EC-Lab (Bio-Logic, sp-200, Seyssinet-Pariset, France) and a CHI 660C potentiostat (CH Instruments Inc., Austin, TX, USA), respectively. For EIS analysis, a bias voltage of 10 mV was applied between WE and CE over a fre-

Morphological Characterization of Immunosensor Surface
In each step of the immunosensor assembly, the surface morphologies of the electrodes were analyzed over a 10 × 10 µm area by atomic force microscopy (AFM; VEECO CP II, Veeco Instruments Inc., New York, NY, USA). The imaging process was operated in contact mode, and scanned at a rate of 1.0 Hz. To show the capability for cis P-tau interaction with the immobilized antibody, the modified electrode was subjected to incubation with the cis P-tau solution in PBS (1.0 × 10 −12 M) for 30 min at RT, followed by washing with 5 mL of PBS before imaging.

Electrochemical Characterization of Immunosensor
Electrochemical impedance spectroscopy (EIS) and CV were used to monitor the electrochemical performances of immunosensor at different steps of the layer-by-layer procedure of immunosensor fabrication. The electrochemical measurements were conducted in 10 mM K 4 Fe(CN) 6 /K 3 Fe(CN) 6 (1:1 ratio) by using a three-electrode set up including the modified Au electrode (1.5 mm 2 surface area) as working electrode (WE), a Pt wire as an auxiliary electrode (AE), and Ag/AgCl as the reference electrode (RE). The EIS and CV were carried out using an EC-Lab (Bio-Logic, sp-200, Seyssinet-Pariset, France) and a CHI 660C potentiostat (CH Instruments Inc., Austin, TX, USA), respectively. For EIS analysis, a bias voltage of 10 mV was applied between WE and CE over a frequency range of 0.1-1000 Hz. The generated EIS data were fitted to the Randles equivalent circuit model using ZView software (Solartron Analytical, Farnborough, UK).

Measurement of cis P-tau in PBS and Human Serum
To investigate the electrochemical response of fabricated immunosensor to the different concentrations of cis P-tau, the differential pulse voltammetry (DPV) was conducted in 10 mM K 4 Fe(CN) 6 /K 3 Fe(CN) 6 (1:1 ratio) in PBS at pH 7.5. Different solutions of cis P-tau in PBS or undiluted human serum with successive concentrations (0 × 10 −14 M to 3.0 × 10 −9 M) were prepared. The human serum was collected from clotted normal blood and was used fresh. Prior to the analysis, each cis P-tau solution was dropped onto the WE surface, incubated at RT for 30 min, and subsequently washed with PBS.

Preparation of Real Human Samples
The written informed consent was obtained from all patients and the sample collection protocol was approved by the Ethics Committee of Royan Institute. After the initial survey including mini-mental state examination (MMSE) evaluation, patients underwent CSF sampling. The CSF samples were drained using 20-gauge yellow lumbar puncture needle inserted into the subarachnoid space at the L3-4 or L4-5 interspace after sterile preparation. The 2 to 5 mL of CSF was then collected in sterile plastic tubes for evaluation of biomarker (CSF cis P-tau) using an in-house ELISA and the immunosensor. Similarly, for the control group, the CSF sample was drained using the same method at the time of spinal anesthesia and subjected for evaluation of cis P-tau. Blood samples were collected from the AD patients at different stages and healthy controls in collaboration with Sasan hospital of Tehran, Iran. Serum samples were derived from the clotted blood and were analyzed fresh. The analysis was carried out blindly according to the clinical status.

Measurement of cis P-tau in Real Samples
The CSF samples were diluted 1/100 in 0.1 M PBS prior to analysis and the serum samples were analyzed without any pretreatment. For immunosensor measurement, each sample was dropped onto the WE surface, incubated at RT for 30 min, washed with PBS, and then, DPV was conducted in 10 mM K 4 Fe(CN) 6 /K 3 Fe(CN) 6 (1:1 ratio) in PBS at pH 7.5. To compare with the immunosensor, an in-house ELISA was produced for the detection of cis P-tau in real human CSF and serum samples. In brief, 2 µg of anti cis-phosphorylated Thr231-Tau diluted in 100 µL of PBS (0.01 M, pH 7.2) and was immobilized in 96-well ELISA plates and stored at 4 • C overnight. To reduce nonspecific binding, wells were rinsed with 5 × 300 µL Tris-buffered saline/Triton X-100 (TBST) using an automatic plate washer and blocked for 45 min at 37 • C with a blocking solution (TPBS + 0.5 mM BSA). Following additional automated washing, 100 µL/well of serial standards or human serum were added and incubated at 37 • C for 1 h. Following an additional wash step, 100 µL of HRPconjugated goat anti-mouse IgG diluted 1:500 in BSA was added to each well, incubated for 1 h at 37 • C and washed. The colorimetric reaction was initiated upon the addition of 200 µL of ready-to-use TMB substrate (Seramun Diagnostica GmbH, Germany), and the plates were allowed to rest for 20 min at 37 • C in the dark. Finally, the reaction was stopped with 100 µL/well of 2 N sulfuric acid, and the absorbance of the samples was determined on a microplate reader at 450 nm (Thermo Fisher Scientific, Waltham, MA, USA). The standard curve was prepared by serial dilution of cis P-tau peptide (cis-phosphorylated Thr231-Dmp-Tau (KVAVVRpT (5,5-dimethyl-L-proline) PKSPS) in PBS ranging from 0.0001 µg/mL to 3 µg/mL. The ELISA assay was performed in triplicate experiments and compared with the results obtained using the immunosensor.

Statistical Analysis
All statistical analyses were done using Student s t-test and p < 0.05 was considered to be statistically significant.

Fabrication and Characterization of Immunosensor
The topographical changes of the electrode surface during the multi-step procedure of the immunosensor fabrication were monitored using AFM. Figure 2 shows the AFM 3D images at the consequent stages of the sensor fabrication. After deposition of the NHSactivated thiol linkers on the GE, the roughness of the electrode surfaces decreased slightly from 142 nm to 135 nm (Figure 2A,B). Upon addition of the macromolecular agent of the cis P-tau antibody, the surface roughness increased dramatically to 144 nm ( Figure 2C), which is similar to that observed in other studies [58,59]. Moreover, the addition of analyte, cis P-tau produced a small change in the surface roughness from 144 nm to 148 nm ( Figure 2D). The AFM data could confirm the successful deposition of the agents at each stage.
slightly from 142 nm to 135 nm (Figure 2A,B). Upon addition of the macromolecula agent of the cis P-tau antibody, the surface roughness increased dramatically to 144 nm ( Figure 2C), which is similar to that observed in other studies [59,60]. Moreover, the ad dition of analyte, cis P-tau produced a small change in the surface roughness from 14 nm to 148 nm ( Figure 2D). The AFM data could confirm the successful deposition of th agents at each stage. EIS and CV were used to confirm the layer-by-layer deposition of substances on th gold electrode surface during the multi-step fabrication process ( Figure 3). EIS is a pow erful instrument for surface characterization and monitoring changes to the interface The EIS data were showed through Nyquist complex-plane diagrams ( Figure 3A). I these diagrams, the semicircle diameter of each impedance spectrum represents the in terfacial charge-transfer resistance (Rct) at each step of the immunosensor construction At the first step, chemical modification of gold surface with MUA/MPA resulted in remarkable increase in the Rct (1.53 to 7.08 kΩ), which could be related to the lower su face concentration of the probe due to the existence of long carbon chains in the MUA MPA layer, which could limit the diffusion of Fe(CN)6 3−/4− redox couple towards th electrode surface. Additionally, the peak shift is an indication of the enhancement of di ficulty of electron transfer, which was probably caused by the negative charge of th electrode surface via the formation of R-COOlayer. This behavior was realized by a re EIS and CV were used to confirm the layer-by-layer deposition of substances on the gold electrode surface during the multi-step fabrication process ( Figure 3). EIS is a powerful instrument for surface characterization and monitoring changes to the interfaces. The EIS data were showed through Nyquist complex-plane diagrams ( Figure 3A). In these diagrams, the semicircle diameter of each impedance spectrum represents the interfacial chargetransfer resistance (R ct ) at each step of the immunosensor construction. At the first step, chemical modification of gold surface with MUA/MPA resulted in a remarkable increase in the R ct (1.53 to 7.08 kΩ), which could be related to the lower surface concentration of the probe due to the existence of long carbon chains in the MUA-MPA layer, which could limit the diffusion of Fe(CN) 6 3−/4− redox couple towards the electrode surface. Additionally, the peak shift is an indication of the enhancement of difficulty of electron transfer, which was probably caused by the negative charge of the electrode surface via the formation of R-COO − layer. This behavior was realized by a remarkable decrease of the current intensity in CV diagram of the electrode modified with MUA/MPA compared to the bare electrode ( Figure 3B). Subsequently, treatment of the MUA/MPA-modified electrode by EDC/NHS showed a declined R ct (2.16 kΩ) and an increased current intensity in CV ( Figure 3B). This could contribute to the elimination of the negative charges through NHS ester formation on the carboxylic groups, which generally reduces the negative charges of the electrode surface and facilities the probe diffusion ability. These results confirmed that the CO 2 groups of MUA/MPA were activated with NHS coupling. Upon the immobilization of cis P-tau mAb on the activated gold electrode, a remarkable increase in the R ct (3.28 kΩ) and decrease in the current intensity ( Figure 3B) were observed due to the binding and accumulation of the macromolecular antibody on the electrode surface which may cause more blockage in the diffusion path of the probe ions towards the electrode surface. The results of EIS and CV were in agreement and confirmed the success of the layer-by-layer assembly process during fabrication and performance of the immunosensor. Upon addition of cis P-tau solution onto the electrode surface, the peak current was further decreased, suggesting the successful recognition of cis P-tau by the antibody and accumulation of the cis P-tau macromolecules on the electrode which can hinder electron transfer between the electrode surface and the solution containing the [Fe(CN) 6 ] 3−/4− redox probe. All the measurements were conducted under pH 7.5, which was determined as the optimal pH of medium for maximal electrochemical signal production in response to cis P-tau ( Figure S1). erally reduces the negative charges of the electrode surface and facilities the probe diffusion ability. These results confirmed that the CO2 -groups of MUA/MPA were activated with NHS coupling. Upon the immobilization of cis P-tau mAb on the activated gold electrode, a remarkable increase in the Rct (3.28 kΩ) and decrease in the current intensity ( Figure 3B) were observed due to the binding and accumulation of the macromolecular antibody on the electrode surface which may cause more blockage in the diffusion path of the probe ions towards the electrode surface. The results of EIS and CV were in agreement and confirmed the success of the layer-by-layer assembly process during fabrication and performance of the immunosensor. Upon addition of cis P-tau solution onto the electrode surface, the peak current was further decreased, suggesting the successful recognition of cis P-tau by the antibody and accumulation of the cis P-tau macromolecules on the electrode which can hinder electron transfer between the electrode surface and the solution containing the [Fe (CN) 6] 3-/4-redox probe. All the measurements were conducted under pH 7.5, which was determined as the optimal pH of medium for maximal electrochemical signal production in response to cis P-tau ( Figure S1).

Selectivity of Immunosensor
To investigate the selectivity of the immunosensor to cis P-tau, the possibility of an off-target response to trans P-tau and major plasma proteins including albumin, beta-2microglobolin, immunoglobulin G, and hemoglobin was studied by DPV analysis (Fig-Figure 3. Electrochemical monitoring of the layer-by-layer assembly process carried out for fabrication of immunosensor, including the cleaned bare gold (blue), chemical modification with linkers MUA/MPA (red), activation of linkers with EDC/NHS (green), immobilization of cis P-tau mAb (Purple), and deposition of cis P-tau (orange). (A) Electrochemical impedance spectroscopy (EIS). The imaginary component of EIS (Z") was plotted against the real component of EIS (Z ) in a Nyquist plot (resistance of solution, (R sol = 33 Ω). (B) Cyclic voltammetry (CV) voltammogram. The electrochemical measurements were conducted in PBS (pH 7.5) containing 10 mM K 4 Fe(CN) 6 /K 3 Fe(CN) 6 (1:1 ratio) by using a three-electrode set up including the modified Au surface as working electrode, a Pt wire as an auxiliary electrode, and Ag/AgCl as the reference electrode.

Selectivity of Immunosensor
To investigate the selectivity of the immunosensor to cis P-tau, the possibility of an off-target response to trans P-tau and major plasma proteins including albumin, beta-2microglobolin, immunoglobulin G, and hemoglobin was studied by DPV analysis (Figure 4). These proteins were chosen as major off-targets because trans P-tau is the other isomer of P-tau, which is not considered as early driver of AD, albumin and beta-2-microglobolin are abundant in CSF and serum and beta-2-microglobolin is routinely tested as a representative of the heavy inflammatory marker proteins in CSF, IgG is an immunoprotein that is elevated in CSF in cases of infection, and hemoglobin is routinely tested as a blood protein [60][61][62][63][64]. The presence of these components in biological fluids may interfere with detection of low abundant biomarkers in clinical samples [65]. The immunosensor response to these proteins was analyzed by DPV method (Figure 4A), and the current intensity change upon incubation with each protein was measured from five independent experiments ( Figure 4B). The data showed that upon incubation with the off-target proteins, the recorded signal was similar to the blank PBS, while a sharp signal rise was observed with the target protein cis P-tau even at lower concentration. The result affirmed that no considerable non-selective binding occurred between anti-cis P-tau antibody and the off-target proteins and the produced signal with incubation of cis P-tau was the result of specific antibodyantigen interactions. This specificity against the cis isomer of P-tau was further confirmed by analysis of signal production in response to higher concentrations of trans P-tau (100, 1000, and 2000 pM), which showed no significant signal change at all the levels of this off-target isomer ( Figure S2). immunosensor response to these proteins was analyzed by DPV method (Figure 4A), and the current intensity change upon incubation with each protein was measured from five independent experiments ( Figure 4B). The data showed that upon incubation with the off-target proteins, the recorded signal was similar to the blank PBS, while a sharp signal rise was observed with the target protein cis P-tau even at lower concentration. The result affirmed that no considerable non-selective binding occurred between anti-cis P-tau antibody and the off-target proteins and the produced signal with incubation of cis P-tau was the result of specific antibody-antigen interactions. This specificity against the cis isomer of P-tau was further confirmed by analysis of signal production in response to higher concentrations of trans P-tau (100, 1000, and 2000 pM), which showed no significant signal change at all the levels of this off-target isomer ( Figure S2).  6 (1:1 ratio) was analyzed by DPV method and (B) the immunosensor specificity was measured by relative signal intensity with respect to the baseline (%IR), %IR = (IBare − IProtein)/IBare × 100, where IBare represents the current intensity of the blank immunosensor and IProtein represents the current intensity of the immunosensor upon incubation with each protein (n = 5). The larger the relative current signal difference (%IR), the greater the protein recognition.

Characterization of the Immunosensor Response to cis P-tau in PBS and Human Serum
After exploring and determining various optimal experimental conditions, the analytical performance of this immunosensor was evaluated by DPV measurements. Firstly, the matrix effect on the current response of immunosensor was studied by analyzing different blank matrices, PBS, serum, and CSF. The current responses were acquired upon  6 (1:1 ratio) was analyzed by DPV method and (B) the immunosensor specificity was measured by relative signal intensity with respect to the baseline (%I R ), %I R = (I Bare − I Protein )/I Bare × 100, where I Bare represents the current intensity of the blank immunosensor and I Protein represents the current intensity of the immunosensor upon incubation with each protein (n = 5). The larger the relative current signal difference (%I R ), the greater the protein recognition.

Characterization of the Immunosensor Response to cis P-tau in PBS and Human Serum
After exploring and determining various optimal experimental conditions, the analytical performance of this immunosensor was evaluated by DPV measurements. Firstly, the matrix effect on the current response of immunosensor was studied by analyzing different blank matrices, PBS, serum, and CSF. The current responses were acquired upon the addition of cis P-tau with successive concentration (5.0 × 10 −14 M to 3.0 × 10 −9 M) to PBS or human serum ( Figure 5). Upon addition of cis P-tau, the peak current decreased compared with the blank in a concentration-dependent manner ( Figure 5A,B). The current response suppression at different concentrations of cis P-tau was calculated from the DPV analysis and plotted in Figure 5C,D. A linear relationship was found between the current change and the cis P-tau concentration at the range of 5.0 × 10 −14 M to 5.0 × 10 −10 M (insets in Figure 5C,D), with the correlation coefficients more than 0.99 for both PBS and serum samples. At a SNR ≥ 3, the limits of detection (LOD) were calculated to be 2.0 × 10 −14 M and 5.0 × 10 −14 M, for PBS and human serum samples, respectively. The obtained LOD was comparable with the electrochemical sensors which have been developed previously for tau protein [17,29,66]. These observations evidenced high sensitivity of the immunosensor for measurement of cis P-tau, either in PBS or a complex matrix such as serum. Figure 5C,D), with the correlation coefficients more than 0.99 for both PBS and serum samples. At a SNR ≥ 3, the limits of detection (LOD) were calculated to be 2.0 × 10 −14 M and 5.0 × 10 −14 M, for PBS and human serum samples, respectively. The obtained LOD was comparable with the electrochemical sensors which have been developed previously for tau protein [17,29,67]. These observations evidenced high sensitivity of the immunosensor for measurement of cis P-tau, either in PBS or a complex matrix such as serum.

Clinical Applicability of the Immunosensor for Analysis of Real Samples
To investigate the applicability of our immunosensor in real clinical settings, we used it to detect cis P-tau in CSF and serum samples collected from human patients. The cis P-tau in real human CSF of healthy and different stages of AD (MCI and dementia) could be detected by the immunosensor in good agreement with the ELISA assay (Table 1). In addition, the standard addition method was used to investigate the sensitivity of the immunosensor to linear changes of the analyte level in the real sample [67,68]. To this end, cis P-tau was added to the real CSF samples with subsequent concentrations and the produced DPV signal was acquired (Table S1 and Figure S3). The added standards were recovered in the range from 97.9 to 106.3% with a high repeatability (RSD < 5.5%), showing the acceptable sensitivity of the immunosensor for detection of small changes of cis P-tau level in real samples. In clinical practice, blood samples are more preferable than the CSF samples that are obtained invasively from the patients, especially when a frequent screening program is desired for early detection among high-risk candidates. Although classic approaches such as ELISA can be established to detect cis P-tau in CSF (cut-off~10-20 pM) by using our mAb, their sensitivity and selectivity is much lower to determine the extremely lower levels of P-tau in complex samples of blood serum [53]. In our experiences with the in-house ELISA, serum detection was not possible at any stages of AD (data not shown). Moreover, ELISA is a label-based technique with clinical applicability that is challenging due to the costly and time-consuming step of analyte labeling with the signal tags. However, the label-free electrochemical detection mechanism of the immunosensor allows for easy, fast, and cost-effective analysis which potentiate its clinical applicability. The immunosensor measurement of cis P-tau in the serum samples of healthy and Alzheimer's subjects at different stages of AD successfully detected cis P-tau at higher levels in AD samples compared with the healthy samples (Table 2). Notably, the cis P-tau was detected at levels correlated with the disease stages which suggests the applicability of the immunosensor for serum detection of AD, even at the earlier stages. This label-free detection mechanism allows for easy, fast, and cost-effective analysis without the need for labeling the analyte with the signal tags that are common in classic methods such as ELISA. In order to evaluate the stability of the immunosensor during long-term storage in clinical practice, the immunosensors were stored at 4 • C and their responses to cis P-tau (10 pM) were recorded over a period of 90 days ( Figure S4). The results showed a slight attenuation trend during storage, so that the amount of current produced on day 90 decreased by only 3.5% compared to the first day. Therefore, the immunosensor can support an acceptable shelf-life which is crucial for application in clinical services.

Conclusions
In this study, we utilized a proprietary antibody against cis P-tau to develop a novel electrochemical immunosensor for the detection of cis P-tau as an early biomarker of AD. The immunosensor was fabricated by covalent immobilization of anti-cis P-tau mAb onto a gold electrode surface by using a combination of NHS-activated thiol linkers. By using DPV electrochemical analysis, the cis P-tau was detected in PBS and human serum matrices with subsequent concentrations up to 0.02 and 0.05 pM, respectively. The immunosensor performed successfully in the real situation as the cis P-tau was measured in human pa-tient CSF samples in good agreement with an in-house ELISA and recovered precisely in manually spiked CSF samples. More importantly, the immunosensor detection was successful in human serum samples collected from AD patients at different disease stages with a low cut-off (0.05 pM), while the detection was not possible with the ELISA. The focus on a specific isomer of P-tau, which is the major early driver of AD, is the main superiority of this work than the previous works on AD detection based on tau measurement ( Table 3). As cistausis is a well-known early driver of tauopathy in several neurodegenerative diseases such as AD, this immunosensor may find a unique clinical application in early diagnostic procedures.  Figure S1.  Figure S3. DPV response of the immunosensor to added standard solutions of cis p-tau to human CSF samples obtained from healthy and Alzheimer's individuals at different stages of AD (n = 3); Figure S4. Comparison of immunosensor DPV response to cis p-tau in PBS (10 × 10 −12 M) after immunosensor storage at 4 • C over a 90 days period (n = 9); Table S1. Precision (RSD%) and recovery study of the immunosensor performed by adding standard solutions of cis p-tau to human CSF samples obtained from healthy and Alzheimer's individuals at different stages of AD (n = 3).