2.1.

Subject

For antibody-based magnetoimmunoassay of salivary $\mathrm{A}\beta$ peptides in this study, two groups were examined: 28 putative ADs and 17 normal controls without any cognitive impairment and neuropathological symptoms. For all AD patients, diagnosis was done by clinically well-practiced medical doctors, according to the clinical mental examination and on the basis of the evidence of cognitive dysfunction, interference in daily life, and intellectual damage. All AD cases also performed the mini-mental state examination (MMSE) that has been generally used to identify current cognitive function of patients and would be helpful for AD diagnosis.^31,32 The mean value of MMSE score for the AD patients was 17.3 ($p=7.31$, maximum 28, minimum 3). The AD group was classified into severe and mild cognitive impairment (MCI) stages. The control group was composed of persons who have no family history of AD and the normal cognitive function and intellectuality. MMSE was also performed for the normal group: the mean value of MMSE score for the controls was 25.5 ($p=2.07$, maximum 28, minimum 22).

2.2.

Saliva Collection

Salivary samples were collected from both AD patients and healthy controls. All procedures carried out in this study were approved by the ethical permission, the Human Subjects Committees of the Chosun University. Informed consent was obtained prior to sample collection from all participants. Salivary samples were obtained using sterile centrifuge containers; thereafter, 2% sodium azide solution was added to prevent microbial decomposition.^30,33 In this procedure, saliva samples were achieved under unstimulated condition, which requires resting condition of whole mouth for a while and subsequent salivary drooling.^24,34 Participants were asked to rinse their mouth with purified water before providing saliva samples of approximately 2 to 3 ml into the containers. The samples were immediately frozen at $-80°\mathrm{C}$ until use. The samples were centrifuged at 1500 rpm for 7 min to remove debris in a similar manner that was previously described.^30,35

2.3.

Sandwich Immunoassay on Magnetic Nanoparticles (Antibody-Based Immunoassay)

Carbodiimide-mediated coupling method was used for coupling of amino group of $\mathrm{A}\beta$ primary capture antibodies (monoclonal antibody, mouse, Covance, Dedham, Massachusetts) to the magnetic nanoparticles ($d=\sim 0.2\mu \text{m}$, Chemicell, Berlin, Germany) containing terminal carboxylate groups, which is a binary covalent binding to guarantee a good immobilization of the antibodies on the nanoparticle surface. As graphically described in Fig. 1(a), 20 μl ($1.8\times {10}^9$ beads) of magnetic bead suspension was washed two times with 250 μl of 2-($N$-morpholino) ethanesulfonic acid (MES, pH 5.0, Sigma-Aldrich, St. Louis, Missouri) buffer using a magnetic separator. After second washing, the particles were resuspended in 250 μl of MES buffer containing 5 mg of water soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, Sigma Aldrich) for 5 min at room temperature on a rotating wheel, which will enhance the uniform binding of the capture antibody to the entire bead surfaces at the next step. After an additional two times washing steps with 250 μl of MES buffer, the EDC-treated particles were resuspended in 250 μl of MES buffer. Given that 10 samples at the final step would be prepared at the concentration of $1.0\times {10}^7$ particles per vial, 14 μl of the EDC-treated bead suspension was added to 86 μl of the MES buffer to prepare a 100-μl bead suspension with concentration of $1.0\times {10}^8$ beads. Thereafter, 2 μl of the $\mathrm{A}\beta$ primary monoclonal capture antibodies were conjugated to the EDC-treated beads in the 100-μl suspension [Fig. 1(b)]. The antibody conjugation was performed at a concentration corresponding to eight times the minimum amount suggested by the manufacturer protocol (Chemicell) to effectively cover all the magnetic particles with the antibodies. The mixture was incubated on a rotating wheel for 2 h at room temperature. The capture antibody-conjugated beads were then washed two times with phosphate-buffered saline (PBS, pH 7.4, Sigma Aldrich) mixture including 0.1% bovine serum albumin (Sigma Aldrich), 0.05% Tween-20 (Sigma Aldrich), and 0.05% sodium azide (Sigma Aldrich). A 250 μl of blocking solution (goat serum, Covance) was introduced to the capture antibody-conjugated beads separated by a magnetic separator. After 30 min incubation, the suspension was washed two times with PBS mixture used at the previous step using a magnetic separator, then resuspended in 300 μl of PBS, and divided into 10 individual sample vials ($1.0\times {10}^7$ beads per 30 μl in a vial). Thereafter, the solutions containing antigens were loaded into each vial [Fig. 1(c)]: (1) For calibration of the photon-counting signals measured by PMT, the $\mathrm{A}\beta$ peptide solutions (Amyloid-$\beta$ protein fragment 1-40 or 1-42, 99%, Sigma-Aldrich) diluted to a few specified concentrations within the ranges from 10 to $4000\mathrm{pg}/\mathrm{ml}$ were introduced into each corresponding vial. A 1-μl drop of the $\mathrm{A}\beta$ calibration solution included the corresponding concentrations. (2) For salivary $\mathrm{A}\beta$ peptide detection for both probable AD patients and normal controls, 30 μl of individual saliva samples collected from the participants were added to the different vials containing capture antibody-treated nanobeads. After 2 h reaction between antigens and capture antibodies at room temperature, all individual vials were positioned on a magnetic separator to be washed two times with PBS mixture, and the $\mathrm{A}\beta$-conjugated particles in each vial were resuspended in 30 μl PBS. A 2-μl drop of solution containing $\mathrm{A}\beta$ primary detection antibodies (polyclonal antibody, rabbit, Invitrogen, Grand Island, New York) was then introduced into each individual vial followed by 2 h reaction [Fig. 1(d)]. The epitopes of $\mathrm{A}\beta$ peptides react with paratopes of the detection antibodies which, at the next step, would bind to secondary antibodies (Q-dots 655, Invitrogen) for quantification by counting the number of photons emitted from Q-dots [Fig. 1(e)]. After 1 h incubation at room temperature, the fluorescence-treated nanobeads were washed with PBS mixture and resuspended in PBS at the final concentration ($1.0\times {10}^7$ beads per 300 μl in a vial). The nanobeads were then stored in a dark place at 4°C until use. To minimize nonspecific binding of the antibodies, we carried out enough washing processes between each step. The magnetic bead assemblage and sandwich immunoassay procedures are schematically described in Fig. 1.

2.4.

Controllable Positioning of $\mathrm{A}\beta$-Conjugated Nanobeads by Magnetic Attraction

Following the consecutive processes of sandwich immunoassay, a 1-μl drop (concentration at $\sim 3.5\times {10}^4$ beads) of the solution containing $\mathrm{A}\beta$-conjugated magnetic nanobeads was gently introduced using a pipette on each sensing spot placed on one end of a steel bar that is in contact with a magnet at the other end. As described in Fig. 1, each sensing area is surrounded by an obtuse-angled arc-shaped PDMS guide and aligned with center of the steel bar with the strong magnetic attraction. We empirically realized that PDMS microfluidics was not a proper material for the treatment of nanoparticles due to a little sticky nature of PDMS that usually caused a tremendous loss of the nanoparticles during the flow, so we could not control the number of nanoparticles (or the size of the circular assemblage) at the final stage of the experiments. With no use of any microfluidic channel, we tried to use a small well ($d=1\mathrm{mm}$) to confine the nanoparticle solution, but it was difficult to fill up the well with nanoparticle solution due to the trapped air inside the well. Therefore, an obtuse-angled arc-shaped PDMS guide was the most proper structure in confining the solution and controlling the formation of the circular assemblage, because its open wall helped the solution being easily filled in the structure. To prevent widespread scattering of nanobeads out of the sensing area and to achieve effective deposition within the area, the introduced nanobeads were required to decrease sedimentation rate with the assistance of hydraulic resistance in the water droplet, which was previously dropped on the sensing area. The magnetic nanobeads in the sensing area gradually formed a circular monolayer assemblage by the strong magnetic attraction from the tiny cylindrical wire, as shown in the schematically enlarged description and the inset. Figure 2 shows the arrayed sensing spots with five consecutive nanobead assemblages and their fluorescent images with which the samples from five different patients would be analyzed at a time. In almost every case of experiments, the size of the magnetic bead assemblages was pretty well adjusted in a controlled manner with a diameter of $\sim 300\mu \text{m}$ on average, and the assemblages were well aligned within each sensing area. It means that the number of magnetic beads in the assemblage was almost consistent and estimated to be approximately $\sim 1.0\times {10}^4$. There seemed to be no direct association between the diameter of circular assemblage and the size of the arc PDMS. The diameter of circular assemblage was affected by the number of nanoparticles existing in a droplet after the solution preparation processes, and also fast sedimentation caused the dispersion of the nanoparticles out of the sensing area.

Fig. 2

The arrayed sensing spots with five consecutive nanoparticle assemblages and their fluorescent images. The magnetic nanoparticles in the sensing area gradually formed a circular monolayer assemblage by the strong magnetic attraction from the tiny cylindrical wire placed beneath each sensing spot. The size of the magnetic bead assemblages was well adjusted in a controlled manner, and the assemblages were well aligned within each sensing area.

2.5.

Photon Counting and Fluorescence Imaging

Quantitative evaluation for salivary $\mathrm{A}\beta$ levels of the AD and control groups was performed by photon counting using a PMT. After a droplet of $\mathrm{A}\beta$-conjugated nanobead solution was supplied on each sensing spot of the array system placed on the stage of the fluorescence microscope (Olympus, Daejeon, Korea, BX51) equipped with the PMT, the nanobead assemblage was formed [Fig. 3(a)], and the excitation light at a wavelength of 480 nm was applied to the assemblage on the sensing area. We measured the number of photons emitted at 655 nm from the fluorescent Q-dots labeled with $\mathrm{A}\beta$ peptides, which was carried out three times and took the average value for each sample. The virtual region-of-interest (ROI) for PMT analysis was set as a $100\times 100{\mu \text{m}}^2$ region on the fluorescent image, whose fluorescent light passes through on the way toward PMT, as shown in Fig. 3(b). The horizontal and vertical slits of the PMT for control of the amount of incident beam were precisely adjusted to the dimension of the squared ROI, meaning that we counted the photons emitted from the nanobeads exactly within the ROI, not from the surroundings out of the ROI. Fluorescence imaging was achieved using a high-sensitive CCD camera equipped to the fluorescence microscope. The power of excitation light from the UV source was fixed at 2.8 mW for all the experiments. In addition, transmission electron microscopy (TEM) images were taken to verify the labeling of Q-dots with $\mathrm{A}\beta$ peptides covalently bound to nanobead surfaces. For the AD case, much more Q-dots were indicated by white arrows in Fig. 3(c) compared with the control case in Fig. 3(d).

Fig. 3

(a) Formation of a salivary $\mathrm{A}\beta$-conjugated nanoparticle assemblage with a diameter of $\sim 300\mu \text{m}$. The number of magnetic beads in the assemblage was almost consistent and estimated to be approximately $\sim 1.0\times {10}^4$. (b) The virtual region-of-interest (ROI) for photomultiplier tube (PMT) analysis was set as a $100\times 100{\mu \text{m}}^2$ region on the fluorescent image, whose fluorescent light passes through on the way toward PMT. The number of photons emitted from the Q-dots was measured three times by a PMT, and the average value was taken for each sample. (c) Transmission electron microscopy (TEM) images were taken to verify the labeling of Q-dots covalently bound to nanobead surfaces for the Alzheimer’s disease (AD) case, showing that much more Q-dots were indicated, (d) compared with the control case.

3.1.

Calibration of PMT Photon-Counting Signal

Prior to the detection of the salivary $\mathrm{A}\beta$ peptides collected from probable AD patients using antibody-conjugated magnetic nanobead immunoassay, we were required to generate two standard calibration curves for the PMT photon-counting signals induced from the fluorescent Q-dots labeled with two different $\mathrm{A}\beta$ species ($\mathrm{A}{\beta }_{40}$ and $\mathrm{A}{\beta }_{42}$). As previously described in brief, each $\mathrm{A}\beta$ species was diluted to the specified concentrations ranging from 10 to $4000\mathrm{pg}/\mathrm{ml}$. A 1-μl drop of each $\mathrm{A}\beta$ species containing different concentrations was added to the capture antibody-treated nanobeads, followed by several processes of incubation and washing. Through the further processes such as detection–antibody conjugation and secondary Q-dot labeling, a certain amount of nanobeads was placed on the center of sensing area using magnetic attraction. The photons of fluorescence emitted from the Q-dots only within the squared ROI were counted by PMT for all calibration solutions of each $\mathrm{A}\beta$ species. Experiments were repeated more than three times for each concentration and for both $\mathrm{A}\beta$ species. The results are described in average values (squares) with standard deviation (SD), as shown in Figs. 4(a) and 4(b), forming a linear configuration as a function of the $\mathrm{A}\beta$ concentration. The fluorescent images of Q-dots for different concentrations (unit: $\mathrm{pg}/\mathrm{ml}$) of $\mathrm{A}{\beta }_{40}$ peptides were described in Fig. 5(a), indicating that a higher concentration of $\mathrm{A}\beta$ species allowed the detecting antibody and Q-dots to be more abundantly conjugated with $\mathrm{A}\beta$ peptides, which generate a stronger emission from a larger number of Q-dots than a lower concentration of $\mathrm{A}\beta$. The dimension of the image indicates that of the ROI.

Fig. 4

PMT measurements of the photon-counting signals induced from the fluorescent Q-dots labeled with two different $\mathrm{A}\beta$ species: (a) for $\mathrm{A}{\beta }_{42}$ and (b) for $\mathrm{A}{\beta }_{40}$. Standard calibration curves (squares) and the photon-counting measurements for AD severe group (red square), mild cognitive impairment (MCI) group (blue square), and control group (green square).

Fig. 5

Fluorescent images of Q-dots at different concentrations. Higher concentration of $\mathrm{A}\beta$ species allowed the detecting antibody and Q-dots to be more abundantly conjugated with $\mathrm{A}\beta$ peptides, generating stronger emission from larger number of Q-dots than lower $\mathrm{A}\beta$ concentration. (a) Standard $\mathrm{A}\beta$ calibration solution at different concentrations (unit: $\mathrm{pg}/\mathrm{ml}$). Fluorescent images of Q-dots conjugated with salivary $\mathrm{A}\beta$ obtained (b) from normal group, (c) from MCI case, and (d) from severe AD case.

3.2.

Salivary $\mathrm{A}\beta$ Levels

The photon-counting assessments for salivary levels of both $\mathrm{A}{\beta }_{40}$ and $\mathrm{A}{\beta }_{42}$ peptides using PMT were performed for totally 45 putative AD patients and controls. After antibody-conjugated magnetic nanobead immunoassaying, it was unequivocal that our method was able to identify the existence of the $\mathrm{A}\beta$ peptides in human saliva as demonstrated in a previous study.³⁰ A sensitive ELISA (Covance) was used as a gold-standard reference using saliva samples from the same participants. The putative AD patients were classified into two groups: severe and MCI stages. They had similarity in age and gender.

The salivary $\mathrm{A}{\beta }_{42}$ levels resulting from the photon-counting measurements showed a distinct inclination to increase for AD patients (red square) compared with control group (green square), as shown in Fig. 4(a), which statistically seemed to be significantly different. Comparison within the AD group also had a tendency toward an obvious increase in the $\mathrm{A}{\beta }_{42}$ level for the severe stage (red square) compared with the MCI stage (blue square), meaning a statistically noteworthy difference, however, which still showed a high SD. This finding showed an antithetical tendency to a previous report, in which the $\mathrm{A}{\beta }_{42}$ levels of severe AD stage were lower than those of more moderate stages.³⁰ Regarding the photon counting of the salivary $\mathrm{A}{\beta }_{40}$, the results showed a similar trend to those of $\mathrm{A}{\beta }_{42}$ cases for all categories of this study, as shown in Fig. 4(b), however, statistically, with no significant differences. These findings also had conflicting results over the previous report in which $\mathrm{A}{\beta }_{40}$ levels remained unchanged for all AD cases as well as the control group.³⁰ However, our results were carefully compared using a sensitive ELISA. The results obtained from ELISA were compared with those from PMT and showed a similar trend between the two results (data not shown). Given that the definite mechanism of how $\mathrm{A}\beta$ peptides accumulate in saliva has not been clearly determined yet, our results might be carefully worth consideration, which should be clarified through the future study with more participants. The fluorescent images of Q-dots conjugated with salivary $\mathrm{A}\beta$ obtained from normal group were shown in Fig. 5(b). Also, the fluorescent images of Q-dots obtained from the MCI and severe cases were shown in Figs. 5(c) and 5(d), respectively. It is apparent from these images that the fluorescent intensity from the severe cases seems to be much stronger than that of the MCI cases, indicating that the higher $\mathrm{A}\beta$ concentration in saliva of the severe cases allowed the detecting antibody and Q-dots to be more abundantly conjugated with $\mathrm{A}\beta$ peptides.