A New Method to Advance the Early Detection of Alzheimer’s Disease

Article By : Tony Chung, Instant NanoBiosensors

A simple, non-invasive and sensitive blood tests that could pinpoint the AD process well before any cognitive symptoms would allow clinicians to give any potential disease-slowing therapy at a very early point in the disease process.

We face a looming global epidemic of Alzheimer’s disease (AD) as the world’s population ages. Modest advances in therapeutic and preventive strategies that lead to even small delays in the onset and progression of AD can significantly reduce the global burden of this disease.

PET imaging and tests that use cerebrospinal fluid (CSF) can be used to identify Alzheimer’s before dementia develops. But PET imaging is expensive, and collecting CSF is invasive. With the advancement of ultra-sensitive blood-based protein detection technology, a simple, non-invasive and sensitive blood tests that could pinpoint the AD process well before any cognitive symptoms would allow clinicians to give any potential disease-slowing therapy at a very early point in the disease process.

ELISA technology, which has been the industry standard for protein detection for over 40 years, has larger margin of errors. The process may involve as many as over 30 steps and needs two to three hours to complete. It is very time-consuming, limited in dynamic linear range and sensitivity, and requires trained technicians to operate. In 2019, Instant NanoBiosensors (INB) launched their Light Sensing Biomarker Analyzer based on FOPPR sensing technology (Fiber Optic Particle Plasmon Resonance) with higher sensitivity than mainstream devices on the market. The patented FOPPR sensing technology has been cited in more than 1,000 scientific publications in the areas of high unmet medical needs and research interests, and overall number of global patents will be more than 35 this year.

Due to this cutting-edge technology, only 20µL of blood is needed, and the result can be known in minutes with three simple steps. Furthermore, Light Sensing Biomarker Analyzer is so small that it can be placed on desks, allowing the analysis to be finished in a convenient and fast way. This simple and ultra-sensitive blood test that could pinpoint the AD process well before any cognitive symptoms would allow physicians to give any potential disease-slowing therapy, say, employing anti-Nf-L, anti-amyloid, anti-tau, anti-GFAP, anti-inflammatory, and so on, at a very early point in the disease process. Doing so likely will increase the chances of finding an effective therapy.


What developments have been happening in the medical electronics sector? What technologies are in the pipeline? How are technology suppliers helping manufacturers improve their processes? All this, and more, in this month’s In Focus series.


Optical fibers transmit light on the basis of the principle of total internal reflection (TIR) and are widely used in the telecommunication industry. Recently, optical fibers have become an important part of sensor technology. Their use as a probe or as a sensing element is increasing in clinical, pharmaceutical, agricultural, industrial, environmental, and military applications. There are advantages in favor of the use of optical fibers in biosensors, such as excellent light delivery, immunity of electrical interference, easy miniaturization, low-cost, great potential in remote and in-vivo monitoring, and capability of multiplex detection using multiple immobilized reagents or multiple fibers.

In the last two decades, thanks to the advances in nanoscience and technology, alternative strategies for the development of new optical sensing platforms based on the extraordinary optical properties of gold nanoparticles (AuNPs) have received tremendous attention. The extinction band of a AuNP results when the frequency of an incident light is resonant with the collective dipole oscillation of the conduction electrons in the AuNP and is known as particle plasmon resonance (PPR), also known as localized surface plasmon resonance (LSPR). The PPR phenomenon leads to a very strong absorption band and thus is especially suitable for the development of optical sensors. Interestingly, the PPR absorption band is also very sensitive to the change of the surrounding medium of the AuNP, offering a class of label-free optical biosensors by using bioreceptor-functionalized AuNPs to interrogate its biomolecular interaction with its binding counterpart. As such, the plasmon absorption-based biosensors are especially attractive because they employ a simple extinction measurement which just requires a simple optical configuration without using a bulky and expensive spectrophotometer. Despite the advantages of the plasmon absorption-based biosensing approach, the absorbance of a AuNP submonolayer obtained by either the transmission mode with a single pass of light or reflection mode with a double pass of light is low, leading to limited sensing sensitivity in biomolecular detection.

Figure 1 3D schematic illustration of the sensor fiber in the microfluidic channel of a FOPPR biosensor upon excitation of an incident light beam.

To overcome the above limitation, we have developed a Fiber Optic Particle Plasmon Resonance (FOPPR) sensor, which is realized by excitation of gold nanoparticles on an unclad section of an optical fiber to generate PPR, as shown in Figure 1. This sensor utilizes the evanescent wave via the multiple TIR scheme in an optical fiber to increase the absorbance of a submonolayer of AuNPs on the fiber core surface. By the TIR scheme, the optical path length increases and the excitation of guided modes in TIRs drastically increases light/matter interaction, thus it dramatically enhances the signal-to-noise (S/N) ratio in measuring the attenuated light transmitted through the fiber, offering advantages like label-free, real-time, high analytical sensitivity (nM~pM), wide linear dynamic range, easy-to-operate, ease-of-multiplexing, ease-of-miniaturization, low-cost optical configuration, and easy fabrication of inexpensive sensor elements. Since the principle of FO-PPR measurements is based on absorption spectroscopy, the use of the normalized sensor response alleviates the need for precise optical alignments, and hence providing the niche to be developed as a low-cost and portable biosensor. When AuNPs are pre-immobilized on the fiber core surface in FOPPR sensor as shown in Figure 2a, we call this method the direct assay. Such an approach has been applied to quantitative measurement of antinuclear antibodies in sera, cytokines and matrix metalloproteinases in synovial fluids, and orchid viruses in leaf saps, messenger RNA of HLA-B27 from patients with ankylosing spondylitis, and single nucleotide polymorphisms of DNA in blood samples, with results agree quantitatively with clinically accepted methods.

Figure 2 Schematics of FOPPR biosensor based on two approaches: (a) direct assay method; (b) FONLISA method. OF = optical fiber, NP = gold nanoparticle, CP = capture probe, A = analyte, DP = detection probe.

Even though the direct assay using the FOPPR biosensor is very sensitive at the pM concentration levels, its sensitivity still may not be enough for some more challenging problems. Recently, we have developed a novel enhancement method called Fiber Optic Nanogold-LInked Sorbent Assay (FONLISA) to further enhance the sensitivity of FOPPR biosensor by at least 3 orders. The method employs the same FOPPR sensor and the same concept of nanoplasmon absorption via optical fiber evanescent wave excitation, but different chemical arrangement as shown in Figure 2b, where a capture probe (CP) is immobilized at the fiber core surface and a free AuNP-labelled detection probe (NP conjugated to DP) is used to interact with an analyte molecule (A) to form a sandwich-like complex on the fiber core surface, resulting in a limit of detection (LOD) at the femtomolar concentration levels. The FONLISA method has been successfully applied to quantitative measurement of procalcitonin in blood plasma, mercury ion in spiked water, and ampicillin in spiked milk samples, with good accuracy and reproducibility.

Neurologic diseases are increasingly recognized as one of the most prevalent diseases with high burden to the patients, their families, and society. The two most burdensome neurologic diseases in the United States are stroke followed by AD and other dementias. The two leading causes of death from neurologic diseases were AD and other dementias followed by stroke. In another study from 1990-2010, ischemic stroke, traumatic brain injury (TBI), as well as AD and other dementias contribute to 22.4%, 11.2%, and 6.4%, respectively, of total global burden of disease. The burden of almost all neurologic diseases increased between 1990 and 2017, due in large part to the aging population. Recently, biomarkers have been steadily assimilated in clinical routine and clinical trials in neurology and can be used for several purposes: to conduct clinical diagnosis, to evaluate disease risk or prognosis, to assess disease stage and to monitor progression or response to therapy.

Since 1995, the excellent outcome of cerebrospinal fluid (CSF) biomarkers in AD have revolutionized the field in neurology. Unfortunately, sampling of CSF through lumbar puncture is invasive. Hence, development of blood-based biomarkers will further advance the field of neurology. Nevertheless, biomarkers in blood are present at very low concentrations that require the use of ultrasensitive techniques having the analytical sensitivity at the femtomolar concentration levels. Another even less invasive but promising source of biomarkers is saliva, which has been demonstrated as a good source of samples for some neurologic diseases, but again, the concentrations of these relevant biomarkers are extremely low.

AD, known as chronic progressive central nervous system degeneration disease, is the most common cause of dementia with a high prevalence in elder above 60 years old. The population living with AD is predicted to reach approximately 152 million in 2050. The severity of AD has become not only the public health problem but also the social issue. Hence, the prevention of AD is one of the most importance tasks in medical science. The symptoms of AD can change with the progress of AD from mild cognitive impairment (MCI) loss to severe memory loss and eventually lead to fatality due to the damage of basic physiological function.

Current research indicated that the main pathological phenomena are amyloid plaques and neurofibrillary tangles in the brain which ultimately break down the structure of brain. Briefly, amyloid plaques arise when an abnormal clumping of amyloid β (Aβ) peptide which is toxic to the brain tissue especially in their oligomeric form; neurofibrillary tangles are built from the phosphorylated tau protein. The function of tau protein was used to support the structure of microtube which dominates the transportation of nutrients throughout neurons in the brain. As the phosphorylation of tau protein occurs, the tau protein collapses from the microtube and twists into tangles. Furthermore, the structure of microtube also disintegrates and disrupts the transportation of nutrients. With the cooperation of plaques and tangles, the brain tissues are prone to gradual decomposition. Yet, the exact mechanism of AD is still not well understood.

The strategy of early diagnosis of AD is to monitor biomarkers related to AD, such as Aβ, tau protein, and p-tau protein. Currently, the detection of biomarkers for AD was mainly performed by enzyme-linked immunosorbent assay (ELISA) in clinical case. However, this antibody-based approach may suffer from several drawbacks such as insufficient analytical sensitivity, narrow linear range, long analysis time, tedious and labor-intensive, non-specific binding, and costly. The immuno-FONLISA approach will offer opportunities to detect Aβ oligomers, tau, and p-tau181 in blood or even in saliva for early diagnosis of AD. To overcome the drawbacks due to the use of antibodies, such as high cost and batch-to-batch variability, DNA aptamers are the alternative bio-recognition agents that may remedy the disadvantages inherited by antibodies as they can be produced by chemical methods which are cheaper and quality assured. In this regard, aptamer-based FONLISA approach may offer a possibility to provide cheaper and more reproducible point-of-care testing (POCT) methods for early diagnosis of AD.

In the case of evaluating genetic risk factors for AD, such as APOE genotypes, the genotype is mainly discriminated by detecting the single nucleotide polymorphism (SNP) of APOE alleles, known as rs429358 and rs7412. The application of real-time polymerase chain reaction (PCR) and sequencing which usually coupled with electrophoresis are the most common methods. Nevertheless, these methods inevitably depend on sophisticate instruments and are time-consuming. Our recent development in using the FONLISA method in DNA detection may provide a faster and cheaper solution to SNP detection of such genes.

Recently, we have achieved a LOD of 2.8 fg/mL (0.056 fM) for glial fibrillary acidic protein (GFAP), which is a promising biomarker of several neurologic diseases. To the best of our knowledge, this LOD for GFAP is superior to that by all other methods. Such a superior analytical sensitivity, together with features like rapid detection (£15 min) and low-cost and portable instrumentation, have strongly support the potential of the FONLISA method to become a player in POCT for early diagnosis of AD.

Biomarkers provide opportunities for early diagnosis and disease modifying interventions. Blood biomarkers brings the hope of affordable, accessible dementia diagnosis globally, including to low- and middle income countries.  In the future, INB’s cost-effective ultra-sensitive miniaturized devices can be helpful for not only in early detection but also in disease monitoring after treatment.  As data will be sent to the cloud platform, the goal of real-time health management can be achieved (digital health). Real-time data and analytics allow clinicians and healthcare delivery system to provide the best care at the right time for the right patient toward better quality and better outcomes for patient journey.  INB believes that Integration of nanotechnology into biosensors could benefit the world by moving bioanalysis from laboratory to any point of use.

 

The innovative application discussed in this article is research-based. Light Sensing Biomarker Analyzer is not intended to be used for any medical application, including in-vitro diagnostics.

 

About the author

Tony Chung, CEO & co-founder of Instant NanoBiosensors (INB), has 20 years of extensive experience in the life science industry with cross-ecosystem connecting, management, commercialization and business development. He plays a key role of using Taiwan’s strengths and ecosystem to build their mobile-size devices. INB has already begun to revolutionize the healthcare system to the next level. In accordance with the corporate vision of ‘Light Saves Lives, Tony is dedicated to invest heavily in R&D to create a mutual benefit for the healthcare industries.

 

About Instant NanoBiosensors (INB)

INB is an innovative life science company which has developed ultra-sensitive digital immunoassay platform based on FOPPR (fiber optical particle plasmon resonance) sensing technology and IN-Chip (auto-flowing microfluidic chip) technology for the life science research and in vitro diagnostics markets enables customers to reliably detect protein biomarkers in extremely low concentrations in blood, serum and other body fluids. Through this platform, INB is applying developed biomarkers which have already translated from preclinical safety assessment to clinical utility in the neuroscience markets for life science research, diagnostics and precision health early screening and monitoring.

Learn more at www.instantnano.com.

 

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  1. Chung TONY says:

    Many thanks to EE Times Asia for the invitation.
    We are looking forward to working together with scientists, clinicians and biopharma companies.
    Tony Chung, Instant NanoBiosenosors