Towards a universal method for detection of Alzheimer's disease biomarkers based on amyloid beta peptide

The selection of blood markers has been introduced before, but no corresponding test chip has been developed.

Or rather, the detection of these protein biomarkers has gradually lost its difficulty and is no longer the forefront of innovation.

This paper, published in 2017-2018, focuses the detection object on specific amyloid peptide biomarkers in spinal cord fluid, and proposes a novel magnetic bead based immunoassay method, in which the capture and detection of antibodies grafted on magnetic beads can bind to monomers a β 1 – 40 and a β 1 – 42 simultaneously in a single step, which is of great innovative significance, and was published in sensors and Actuators B-chemical。

Preface

In this study, one-step immunoassay method based on magnetic beads was developed by the problem group of the 11th University of Paris. The method is operated by micro fluid drops, which is used to determine the molecular diagnosis of Alzheimer’s disease (AD), a mature biomarker of amyloid peptide.

The capture antibody (i.e. the N-terminal specific monoclonal anti-A antibody to a β 1 – 42 and a β 1 – 40) was transplanted onto the magnetic beads using the sandwich assay developed.

The 1 – 16 monoclonal antibody labeled with peroxidase of the detection antibody (i.e. amyloid beta protein) could bind to the monomer peptide simultaneously in one step.

The team successfully detected a β 1 – 42 and a β 1 – 40 in cerebrospinal fluid (CSF) samples using the developed batch immunoassay method. In order to extend the traced a β peptide profile beyond a β 1 – 42 and a β 1 – 40 for more accurate diagnosis of AD, the sandwich assay was developed for the first time as a downstream module in combination with peptide fractionation and collection using capillary isoelectric focusing.

The batch immunoassay was reduced to a special microfluidic droplet platform, which greatly reduced the sample volume and increased the flux. Using a series of 4 programmable magnetic tweezers to manipulate a series of nano scale confined droplets containing magnetic beads, samples, washing and detection solutions, eight analysis sequences can be completed in 45 minutes.

This droplet based immunoassay is implemented in a dedicated platform integrated with an internal light-emitting diode (LED) – based fluorescence detector, thus replacing the conventional microscope setup, significantly reducing the construction cost and simplifying the detection scheme. Using this microfluidic configuration and led based detection, information about a β 1 – 42 and a β 1 – 40 concentrations can be collected in a single sequence with less than 1 l of sample.

Highlights

Single step magnetic beads based immunoassay

In the conventional ELISA, several manual operations are needed to achieve the following functions respectively:

The antigen (i.e. a β peptide) was captured on the solid support (i.e. magnetic beads) grafted with capture antibody;

The binding peptide of antibody and immobilized antibody was detected;

In order to make the ELISA microfluidic platform for mass detection, it is necessary to simplify the operation and minimize the error accumulation. Therefore, the research group first developed a single-step intermittent magnetic immunoassay to detect a β 1 – 40 and a β 1 – 42 from standard solutions.

The scheme utilizes two different epitopes on a β peptide that can be used to bind capture antibodies (i.e., 12f4 for the C-terminal of a β 1 – 42 and g2-10 for a β 1 – 40) and detect one of them (6e10-hrp of the N-terminal of a β 1 – 42 and a β 1 – 40). HRP was then used to react with 10-acetyl-3,7-dihydroxybenzoxazine (adhp) in quantared substrate to produce highly fluorescent and soluble resorufin.

Compared with the traditional immunoassay method, the overnight incubation at 4 ° C under mild shaking conditions required much shorter operation time on the new platform and was compatible with microfluidic operation of RT.

CIEF based compartmentalization of a β peptides as afore front of magnetic beads based immunoassays

More effective diagnosis of ad can be achieved by considering a broader a β peptide spectrum other than a β 1 – 42 and a β 1 – 40. In this case, it is necessary to expand the detection of other a β peptides as complementary potential biomarkers of AD, rather than limited to the established peptides (a β 1 – 42 and a β 1 – 40).

However, due to the lack of specific antibodies to all a-peptide targets, immunoassay can not easily achieve this goal. On the other hand, this limitation can be overcome by combining developed immunoassays with pH gradient induced peptide compartmentalization under high electric field (CIEF).

Before immunoassay, the module integrated with this electrical method can separate amyloid protein according to its characteristics, and it can be denatured or oligomerized without organic solvent.

During CIEF, once a peptide reaches the pH corresponding to its PI, it will be focused and concentrated to different regions along the capillary. The first region is a β 1 – 40 (pi = 5.33), the second region is a β 2 – 40 (pi = 5.98), and the last region is a β 5 – 40 (pi = 6.46). Each of these components is then collected to the detection end of the capillary by pressure, and the magnetic bead based immunoassay is performed in a batch manner.

In this case, magnetic bead based immunoassay can be used as an effective, simple and inexpensive tool to visualize peptide segments and monitor collection operations.

gel electrophoresis

Magnetic beads based immunoassays of a β peptides in a microfluidic droplet platform

In order to provide a higher degree of automation, minimize errors caused by manual operation, significantly reduce sample / reagent volume and significantly reduce operation time, the developed batch immunoassay is then transformed into a microfluidic droplet platform to realize the diagnosis of ad at the molecular level.

The system starts with absorbing the droplet flow from the tank, and then delivers the droplet flow to the detector through the electromagnetic tweezers and mixing plate.

Through the on / off trigger of magnetic tweezers, the magnetic beads are transferred from the initial droplet to the sample droplet in the sample incubation step, then transferred to two droplets in the washing 1 and washing 2 steps, and finally distributed to the detection droplet.

In each step, the beads stay in the droplet for a certain period of time (i.e. incubation time), and the droplet flows through a certain distance (i.e. droplet movement distance) in the tube.

In the sample incubation step, beads are dispersed in droplets containing the sample (i.e. peptide) and the antibody to be detected. The residual (nonspecific binding) detection antibodies are then washed from the beads in washing 1 and 2 steps.

Subsequently, in the sample detection step, the washed beads are transferred to the detection droplet containing the substrate for the enzymatic reaction, followed by fluorescence detection.

Deficiency and thinking

Magnetic beads are obviously a high-throughput and highly maneuverable detection method, which is very suitable for the detection of complex targets. The purpose of detecting a β peptide profile is obviously one of its applicable scenarios.

However, I wonder if there are faster methods, such as electrochemical method, to realize the detection mode similar to paper-based microfluidic. That level of fast screen testing is the direction of the future. However, at this stage, this level of testing equipment and enough to update the existing technology to complete some early inspection and quick screening work.

Summary

Using the droplet based platform, the volume required for each droplet operation was significantly reduced from 50 μ L in batch mode to only 200 NL. Therefore, a significant reduction in sample and reagent consumption can be achieved in this drop based immunoassay.

The entire operating time was also significantly reduced to increase the flux, with eight measured sequences performed in less than one hour, rather than two hours in batch mode. In addition, due to the high degree of automation, operator intervention is minimized.

Quote

Mai, T., Ferraro, D., Aboud, N., Renault, R., Serra, M., Tran, N., Viovy, J., Smadja, C., Descroix, S., & Taverna, M. (2018). Single-step immunoassays and microfluidic droplet operation: Towards a versatile approach for detection of amyloid-β peptide-based biomarkers of Alzheimer’s disease. Sensors and Actuators B-chemical, 255, 2126-2135. DOI:10.1016/J.SNB.2017.09.003.

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