Alzheimer's Disease (AD) is a progressive neurodegenerative disorder, for which early diagnosis remains a major challenge. Our objective is to achieve early detection of AD.

We employed SELEX (Systematic Evolution of Ligands by EXponential Enrichment) to screen for aptamers capable of specifically binding to BD-Tau (Brain-Derived tau) protein.

We aim to develop a BD-Tau protein aptamer-based biosensor to measure BD-Tau levels in blood samples. Given its advantages of rapid analysis, low cost, high sensitivity, and ease of integration, we plan to integrate microfluidic technology to develop a solution for the early detection of Alzheimer's disease (AD).

Alzheimer's disease (AD) is a progressive neurodegenerative disorder. Currently, there are approximately 60 million people living with dementia globally (PMC8810394, PMC9588915), with AD accounting for 60%-70% of cases. This number is projected to rise to 153 million by 2050. In China, the number of individuals with AD and other dementias reached 56.85 million in 2021 (Chen etal,2025 ), and the prevalence increases significantly with age .

Current clinical diagnosis of Alzheimer's disease primarily relies on methods such as clinical symptom assessment, neuroimaging examinations, and cerebrospinal fluid (CSF) biomarker analysis. However, each of these methods has its own limitations.

While blood testing is a major research focus, it faces two major challenges: a lack of validated biomarkers and a shortage of automated detection systems.

Although studies have confirmed that BD-Tau can serve as a biomarker for Alzheimer's disease (Gonzalez-Ortiz etal, 2023) and that it is detectable in blood, no convenient detection method is currently available.

Currently popular microfluidic technology, the Microfluidic Chip is a miniaturized platform that utilizes micrometer-sized channels (typically 10–500 μm in width) and functionalized structures to achieve precise manipulation and analysis of fluids(Song etal, 2011)..

The core principle of the chip is to integrate multiple functions of traditional laboratories—such as sample processing, reaction, separation, and detection—into a chip measuring only centimeters or millimeters in size. This approach holds promise for addressing the current challenges in the early diagnosis of Alzheimer's disease .A research team has developed a multivolume barcode chip (V-Chip) that successfully integrates these functions into a single device. The primary strength of the V-Chip lies in its ability to directly present quantitative results in a barcode format on the device itself, eliminating the need for any external equipment.The underlying principle operates by decomposing hydrogen peroxide to generate oxygen, which directly propels the movement of ink within the chip's channels, thereby enabling quantification(Li etal, 2016).

Our project aims to achieve accurate and rapid detection of Alzheimer's disease (AD) through microfluidic technology. The project will first employ the SELEX process to screen for specific aptamers that bind to BD-tau. Subsequently, by integrating these aptamers with microfluidic technology, we will develop a novel detection method for early diagnosis of AD. This innovation provides crucial technical support for developing a new generation of portable rapid AD diagnostic devices, effectively addressing the limitations of traditional laboratory methods such as complex procedures, long processing times, and dependence on large-scale instruments.

Functional Testing of Key Modules

Sample Injection Zone: For loading serum/plasma samples to be tested

Reaction Microchamber: Pre-loaded with H₂O₂ solution as catalytic reaction substrate

Signal Detection Zone: Filled with red-dyed silicone oil as visual flow indicator

Figure 1. Schematic diagram of the chip design.

The microfluidic design is primarily divided into three parts.

Stage l: Target Identification and Signal Activation (Panel A-i)

When the patient’s blood sample is loaded into the first chamber (which contains our patent-pending cleavage assay) it kicks off. In this case, the BD-tau protein target induces an activation of a Cas12aenzyme complex. This activated enzyme subsequently makes a very specific nick in ssDNA linker molecule. This linker's primary role is to physically link the Platinum Nanoparticle (PtNP) (our signal engine) to a magnetic bead. When cleaved, the PtNP is detached from the magnetic bead. The quantity of released PtNPs is linear with the initial content of BD-tau protein.

Stage 2.On-Chip Magnetic Separation (Panel C-ii)

The chip is subsequently translated, causing the fluid to enter the next region of channel. Then, an external magnet is introduced to trap and retain all the magnetic beads (including bearing or not their PtNPs). This is an important purification step: only the liquid with the actually floating relcased PtNPs can pass to the next stage, so that the final signal is rather specific and reliable.

Stage 3.Catalytic Amplification and Visual Readout(Panel C-iii)

The purified PtNP-enriched fluid is guided into a reaction chamber, where it is combined with a premixed reagent H 2O2. The platinum nanoparticles are supercatalysts, quickly decomposing the hydrogen peroxide into gascous oxygen (O2). This reaction will give off a large amount of gas, increasing the pressure in the sealed microchannel. This pressure is functionally equivalent to the piston of a syringe, which moves a pre-loaded colored ink slug along an assigned, calibrated reading channel. The greater the distance travelled by the ink slug, the more oxygen produced(which is directly related to the initial concentration of BD-tau protein). A larger amount of BD-tau causes more ink to move, thus offering an casy-to-see and sensitive test without any electric readers.

The design of microfluidic chips is a critical component in achieving efficient and accurate detection of AD, encompassing multiple aspects such as material selection, structural design, fabrication processes, and system integration, all of which are particularly important.

The material of the chip determines its performance. Chip material selection must adhere to the following principles: good biocompatibility, electrical insulation, and thermal dissipation properties; excellent optical characteristics; a surface amenable to modification; along with simple fabrication processes and low manufacturing costs.

Currently, the primary materials used for fabricating microfluidic chips include single-crystal silicon wafers, quartz, glass, among others, with polymers being the most common. We have selected glass as our material of choice.

The benefits of glass chips include:

Excellent Optical Clarity - High transparency across visible and UV spectra, ideal for optical detection methods.

Superior Chemical Resistance - Withstands most organic solvents and aggressive reagents used in bioassays.

High Thermal Stability - Maintains structural integrity under temperature cycling and high-temperature operations.

Biocompatibility - Minimal nonspecific binding and compatible with biological samples.

Electroosmotic Properties - Naturally suitable for electrophoretic separations.

Rigidity - Maintains channel integrity during fluidic operations.

Fabrication begins with designing fluidic channel layouts in AutoCAD, which are then transferred onto 75 × 50 mm glass slides via standard photolithography and etching techniques to create six parallel microchannels. The final device architecture comprising upper and lower substrates is schematically presented in the accompanying figure 2.

Figure 2.Chip Fabrication

Materials: 

Procedure:

1. Preparation of Aptamer-dsDNA Conjugate

1. Corresponding modified oligonucleotides—10 μM BD-tau aptamer-F, dsDNA-F, and dsDNA-R—were synthesized by a commercial supplier.
2. The three components were mixed at a 1:1:1 molar ratio in a final 1× Taq buffer and thoroughly combined.
3. The mixture was incubated at 95°C for 5 minutes, then gradually cooled to room temperature at a rate of 2°C per minute.
4. The resulting aptamer-dsDNA conjugate was quantified using a NanoDrop spectrophotometer, diluted to a final concentration of 5 μM, and stored at -20°C for future use.

2. Conjugation of PtNP with Aptamer-dsDNA

1. DNA Activation: Commercially available or synthesized thiol-modified DNA (SH-DNA) is activated using a reducing agent such as Tris(2-carboxyethyl)phosphine (TCEP). This step cleaves potential disulfide bonds and reduces the terminal group to a reactive free thiol (-SH)
2. Mixing: The activated thiolated DNA is mixed with the platinum nanoparticle (PtNP) solution to initiate the conjugation process.
3. Aging (Incubation for Conjugation): The mixture is incubated in 10 mM Tris-HCl buffer (pH = 7) for 12 to 16 hours (overnight) to facilitate the complete formation of Pt-S bonds.
4. Salt-Aging: To increase DNA loading density on the PtNP surface without inducing aggregation, a "salt-aging" process is performed. The NaCl concentration is increased stepwise (e.g., from 0.1 M to 0.3 M, then to 0.5 M) by adding small aliquots of a high-concentration salt solution (e.g., 2-5 M NaCl). This gradual increase helps neutralize electrostatic repulsion, allowing more DNA strands to pack densely onto the particle surface. The solution is typically incubated for 30-60 minutes at each intermediate salt concentration.
5. Purification: Finally, the conjugate is purified to remove unbound free DNA. The solution is centrifuged at high speed (e.g., 12,000 - 16,000 x g for 15 - 30 minutes). The supernatant, containing the unbound DNA, is carefully removed, and the pellet of PtNP-DNA conjugates is re-dispersed in a suitable storage buffer (e.g., TE buffer with 0.1 - 0.3 M NaCl). This washing process is usually repeated 2-3 times to ensure complete purification.

3. Preparation of the Aptamer Sensor

1. The streptavidin magnetic beads stored at 4°C were taken out and thoroughly vortexed to ensure a homogeneous suspension.
2. A 20 μL aliquot of streptavidin magnetic beads was transferred into a nuclease-free 1.5 mL centrifuge tube. The tube was placed on a magnetic rack for 1 minute to separate the beads from the storage solution, after which the supernatant was carefully aspirated and discarded.
3. The beads were washed with 200 μL of 1× PBST buffer, vortexed to resuspend, and then magnetically separated for 1 minute. The supernatant was removed. This washing procedure was repeated three times to thoroughly remove the NaN₃ preservative present in the streptavidin magnetic bead storage solution.
4. After washing, 20 μL aliquots of pre-quantified 10 μM biotin-modified Com DNA3, is added to separate centrifuge tubes. Each was then brought to a total volume of 200 μL by adding 1× PBST buffer.
5. The mixtures were vortexed thoroughly and then incubated on a rotator for 30 minutes to allow full binding between the Com DNA and the streptavidin on the magnetic beads.
6. After incubation, the tubes were placed on the magnetic rack for separation. The supernatant was aspirated and discarded to remove any excess Com DNA that did not bind to the beads.
7. The beads were washed with 200 μL of 1× PBST buffer, vortexed, magnetically separated for 1 minute, and the supernatant was discarded. This washing step was repeated twice.
8. After washing, 20 μL of the pre-prepared 5 μM aptamer-dsDNA-PtNP conjugate was added to each centrifuge tube. The volume in each tube was then adjusted to 200 μL using 1× PBST buffer.
9. The mixtures were vortexed thoroughly and incubated on a rotator for 30 minutes to allow complete hybridization between the aptamer-dsDNA-PtNP and the Com DNA.
10. After incubation, the tubes were placed on the magnetic rack for separation. The supernatant was aspirated and discarded to remove any excess unbound aptamer-dsDNA.
11. The beads were washed with 200 μL of 1× PBST buffer, vortexed, magnetically separated for 1 minute, and the supernatant was discarded. This washing step was repeated three times.
12. After the final wash, 900 μL of 1× PBST buffer was added to each tube to resuspend the "magnetic bead-Com DNA/aptamer-dsDNA" complexes in the buffer.

4. Detection of BD-tau

1. Add the blank control, Tau, BSA, and BD-tau at concentrations of 1 μM, 10 μM, and 100 μM to the sample wells.
2. Following a 40-minute incubation, magnetic separation was performed. The glass slide was then advanced, which revealed the test results

We also developed a lab prototype for our TauTrack microfluidics diagnostic platform and tested the its functionality accordingly. Please note that due to issues of time and material (especially the difficulty and time-consuming nature of producing integrated platinum nanoprobes, we chose an alternative using aptamer–comDNA–PtNP. By doing so we can design and experiment with all core functions of the TauTrack “samplein, resultout” microfluidics platform comprised “onchip no human interference” without changing the chip device process.

How to Reconstitute？

A BD‐Tau aptamer is used to bind the PtNP.

The Aptamer contains a short "dock" sequence that will hybridize with a complementary DNA (comDNA).

Streptavidin‐coated magnetic beads are used to capture the biotinylated comDNA. PtNPs are initially fixed to the beads through the aptamer‐comDNA duplex as a complete sensor.

How It Fits the Workflow on‐chip in Three Stages

Stage 1: Target identification and release

BD‐Tau in the sample will bind the aptamer to destabilize the duplex (assisted by a designed toehold) and displace the comDNA.

Results: The PtNP‐aptamer is released into solution and the comDNA remains on the bead. The level of released PtNPs increases as the BD‐Tau concentration rises.

Stage 2: Magnetic separation

A magnet placed externally immobilizes the beads (together with any PtNPs which are still tethered).

Only the supernatant containing truly released PtNPs can pass through to the next chamber.

Stage 3: Catalytic amplification and readout

The released PtNPs cause decomposition of H2O2 to generate oxygen in a closed chamber.

The pressure which is produced pushes a colored ink slug; the distance it moves reflects the level of BD‐Tau.

Figure 3. Prototype of TauTrack Microfluidics Diagnostic Platform of BD-tau

This figure 3 shows the encouraging results of a validation experiment carried out on our microfluidic chip proving its fundamental abilities: high specificity and strong, clear, quantitative, dose-dependent response to our selected target biomarker (BD-tau). The purpose of the experiment was to evaluate the performance of the platform with different protein samples, which were read in calibrated reading channels.

Experimental Setup:

Samples of six different loaded parallel microfluidic channels were collected to verify the performance of the assay:

Control: A buffer solution containing no protein to define the baseline (zero-signal) position of ink.

Channel 1#(BSA): Negative control with Bovine Serum Albumin, a typical non target protein.

Channel 2# (Tau): NTC, a general positive control for specificity against non-brain protein.

Channel 3# (1 μM BD-tau): Desired marker with low concentration.

Channel 4# (10 μM BD-tau): Medium in concentration of the target biomarker,

Channel 5# (100 μM of BD-tau): The high concentration reference biomarker.

Analysis of Results:

In fact the results are a very direct and unambiguous proof of concept for our technology:

High specificity verification: The Control channel and negative control channels 1#(BSA) & 2# (Tau) all have little to no displacement of the red ink slug. This is an important finding, as it demonstrates that our assay is very specific. It does not create a false- positive signal in the existence of other high abundant protein even non-target, closely related forms of Tau protein.

Demonstration of quantitative detection: For channels spiked with target BD-tau protein, it exhibits a representative signal (c.g.,gradually enhanced) positively depends on its concentration.

On the other hand, in Channel 3#, the 1 μM BD-tau sample produced sufficient oxygen pressure to drive the ink slug a substantial length into the reading channel.

On the contrary, in channel 4\#,10 μM of BD-tau sample (magnified by a factor of ten)can make a much stronger response, driving the ink slug far beyond.

In Channel 5#, the effect of BD-tau was most pronounced when a concentration of100 μM was used, as the ink slug travelled almost to the top of calibrated channel. This experiment serves to validate core concepts of our TauTrack integrated microfluidic platform. The see-through, dose-dependent transport of the ink slug demonstrates that it is possible to accurately and quantitatively assess a sample's concentration of BD-tau with our “sample-in, result-out" system. No control channels show any signal, and this lack of off-target is indicative of excellent specificity for our internally developed assay with a high level of confidence. This is a strong proof of concept and shows readiness for additional pre-clinical and clinical advancement of our technology.

Although our experimental results demonstrate the feasibility of integrating microfluidic technology with aptamer sensors, numerous challenges remain. Future efforts could explore novel nanomaterials or surface modification techniques to enhance probe affinity and anti-interference capability. Large-scale experimental data is required to optimize product performance, including parameters such as incubation time and temperature.

Furthermore, under authorized conditions, the utilization of real clinical samples for testing is crucial for product iteration and refinement. While microfluidic technology holds immense potential for the early detection of Alzheimer's disease, significant hurdles must be overcome to translate this technology from the laboratory to widespread clinical application.

Microfluidic technology offers a revolutionary solution for the early detection of Alzheimer's disease, overcoming the key limitations of traditional diagnostic methods such as high subjectivity, substantial cost, and invasiveness. By integrating miniaturized design, automated operation, and highly sensitive detection, microfluidic platforms enable early diagnosis even before the onset of clinical symptoms in AD, creating opportunities for timely intervention and treatment.

Chen HM, Shen K, Ji L, McGrath C, Chen H. Patterns and trends in the burden of Alzheimer's disease and related dementias in China (1990-2021) and predictions to 2040. J Alzheimers Dis. 2025 Jun;105(3):882-892. doi: 10.1177/13872877251333108. Epub 2025 Apr 22. PMID: 40261311.

Gonzalez-Ortiz F, Turton M, Kac PR, Smirnov D, Premi E, Ghidoni R, Benussi L, Cantoni V, Saraceno C, Rivolta J, Ashton NJ, Borroni B, Galasko D, Harrison P, Zetterberg H, Blennow K, Karikari TK. Brain-derived tau: a novel blood-based biomarker for Alzheimer's disease-type neurodegeneration. Brain. 2023 Mar 1;146(3):1152-1165. doi: 10.1093/brain/awac407. Erratum in: Brain. 2023 Oct 3;146(10):e89-e90. doi: 10.1093/brain/awad208. PMID: 36572122; PMCID: PMC9976981.

Huh, D., Matthews, B. D., Mammoto, A., Montoya-Zavala, M., Hsin, H. Y., & Ingber, D. E. (2010). Reconstituting organ-level lung functions on a chip. Science, 328(5986), 1662–1668. https://doi.org/10.1126/science.1188302

Li, Y., Xuan, J., Song, Y., et al. (2016). Nanoporous glass integrated in volumetric bar-chart chip for point-of-care diagnostics of non-small cell lung cancer. ACS Nano, 10, 1640–1647. https://doi.org/10.1021/acsnano.5b07357

Lin, B. C. (2019). Organ-on-a-chip. Science Press.

Song, Y., Zhang, Y., Bernard, P. E., et al. (2012). Multiplexed volumetric bar-chart chip for point-of-care diagnostics. Nature Communications, 3, 1283. https://doi.org/10.1038/ncomms2292