Patent Application
20250020671
The present invention relates to a low-cost, rapid, real-time and simple immuno-optomagnetic point-of-care (PoC) assay. More particularly, the present invention relates to a method for multiplexed detection of a panel of four biomarker proteins in serum for the diagnosis of Alzheimer's disease and it does not require a glass chip platform to generate the signal.
Alzheimer's disease (AD) is an incurable disorder characterized by progressive memory loss, mild cognitive impairment (MCI) to profound cognitive failure. In 2019, World Health Organization (WHO) has recognized AD and dementia as a seventh leading cause of death (worldwide 2.7%) among top 10 leading causes of death in the World. About 65% of deaths from Alzheimer's disease and other forms of dementia are women (https://www.who.int/news-room/factsheets/detail/the-top-10-causes-of-death).
Longer life-expectancies have led to an increased number of neurodegenerative diseases globally [1]. The number of people with Alzheimer's disease or dementia in Turkey alone is reported to be 331,512 in 2012, which represents 0.44% of total population at the time. This number has now increased to 528,547 cases which is about 0.65% of Turkish population according to 2019 reports in the EU's Alzheimer Europe Yearbook L21. In another survey limited to a total 3100 inhabitant population of Eskisehir city in Turkey showed that 51.1% of these inhabitant population were suffering from the most common type of disease was vascular dementia. The same study also found that the remaining 48.8% were found to be suffering from Alzheimer's disease (AD) and 0.1% were with other type of dementias [3, 4]. Turkey's Statistics Institute report suggests that the AD will rise to over 10% in elderly population over age 65 years by 2023 and continue to increase rapidly [5]. The exact reason for the prevalence and risk of dementia is unclear, however Turkey's rapid development and urbanization have been considered to have influence on this disease [6].
In general, much of AD cases have been found sporadic in nature, while there is increasing evidence from the population-based neurological and neuroimaging studies showing that mixed AD and vascular brain pathologies accounts for most of dementia cases in elderly people [7]. AD is linked to multiple risk factors including lifestyle, smoking, alcohol misuse, unhealthy diet that are the sources for a range of diseases that are ultimately linked to dementia.
Current understanding for interventions to prevent the AD or dementia is somewhat disappointing, because the multi-factorial and heterogeneous character of AD is only identified in its late-onset. Timing of AD diagnosis becomes critical especially when the pathology process begins 10-15 years before it shows its symptoms [8]. However, it is difficult to determine the exact time of onset of AD. Lately, there is a shift toward early diagnosis of AD which relies on early identification of significant episodic memory impairment and use of distinctive and reliable biomarkers of AD as revised diagnostic criteria [9].
Earlier standard measures for the diagnosis or clinical manifestations of AD has been through cognitive dysfunction and impairments of activities. It is followed by the neuroimaging using MRI allowing detailed examination of brain structure and hippocampal volume measurement or histopathological identification of amyloid plaques after postmortem [10].
All the existing traditional diagnosis methods fail to address the progression of the Alzheimer's disease even before it precedes or show symptoms in suspected individuals. There is an enormous gap in early diagnosis and monitoring progression of AD in a minimally non-invasive way that remains to be fulfilled.
There is a potential for identifying AD at the very early stages and its progression by a minimally-invasive, easily accessible blood serum/plasma biomarker measure for effective diagnosis, which required extensive research on utilizing a class of AD-biomarkers for PoC tools for early diagnosis. Therefore, detection of AD-biomarkers in blood serum/plasma require standardization before forming core elements of AD diagnostic criteria. Detection of biomarkers in blood serum/plasma, such as amyloid β40/β42, tau protein and BDNF are among the most promising AD diagnostic methods. However, these biomarkers occur in very low concentrations in blood serum/plasma which makes it extremely difficult to precisely and accurately detect such biomarkers.
Most current detection methods rely on a single biomarker for AD diagnosis, which is highly unreliable for accurate AD diagnosis [11]. Clinical standardization of AD diagnosis based on AD biomarker levels in serum/plasma or CSF is needed, which is currently unclear.
Limited attempts have been made thus far in detecting multiple AD biomarkers from a sample that has great potential in enhancing the accuracy of AD diagnosis [8]. Simultaneous measurement of multiple biomarkers present in a sample could provide a high degree of accuracy in diagnosis of AD.
In a few cases, ratio of a pair of biomarkers, such as CSF or blood plasma Aβ42:Aβ40 has been used as indicator for the disease due abnormal Aβ metabolism, and in other cases, increased T-tau and P-tau due to neuronal damage and accumulation of tau have also been used for diagnosis of AD [1]. All these detection methods required laborious proteomics, magnetic resonance or spectroscopic tools for diagnosis that are expensive and time-consuming.
There is ever increasing demand for more accessible and affordable healthcare for chronic as well as infectious diseases. Extension of traditional diagnostic approaches to PoC diagnostics is critically important for affordable, rapid and accurate diagnosis of chronic diseases, such as AD that currently presents significant challenges. PoC based diagnosis offer advantageous of speed, sensitivity, specificity, and compatibility of using them near the patient bedside.
Recent developments in mobile technologies, nanotechnology, imaging systems and microfluidics have transformed the innovative solutions toward developing new, low cost and portable PoC diagnostics [12].
In the last five years, major advancement took place in nanotechnology exploiting the unique physico-chemical properties of nanoparticles, nanocrystals and other nanomaterials. These nano-sized materials are found to have many applications, including in biosensors or bioanalytical assays and their improvement. Nanomaterials have started to use in construction of biosensor based on classical antibodies (immunosensors) that remain the most common recognition elements in research and commercial affinity assays. Novel strategies for sensitive detection of biological responses in healthcare continue to be a priority for PoC applications. The current approaches require improvement toward developing novel multiplexed detection systems and nanomaterials-based research, exploiting the use of multimodal nanoparticles which will contribute to simpler and more sensitive bioanalysis suitable for PoC detection.
Previously, ELISA based methods have been proved to be most sensitive but rely on detection of a lone biomarker at a time, which is time consuming and highly expensive. Simultaneous measurement of multiple biomarkers present in a sample could provide a high degree of accuracy in diagnosis of AD. In a few cases, ratio of a pair of biomarkers, such as CSF or blood plasma Aβ42:Aβ40 has been used as indicator for the disease due abnormal Aβ metabolism, and in other cases, increased T-tau and P-tau due to neuronal damage and accumulation of tau have also been used for diagnosis of AD.
All these existing detection methods required laborious proteomics or magnetic resonance (MR) or spectroscopic tools for diagnosis that are expensive and time-consuming. Many different commercial devices are available for the simultaneous detection of clinical parameters, including electrolytes or acute metabolites. For example, Abbott i-STAT system, Abaxis Piccolo Xpress or Novo StatSensor) or immunoassays such as AQT90 Radiometer and PATHFAST analyzer [13, 14]. There has been expensive and complicated assays performed for detecting varying levels of Aβ42, tau protein and BDNF biomarkers using traditional immunoassay or ELISA based detection, respectively [11], [15]. However, these systems are expensive or bulky bench-top analyzers only capable of detecting limited type of analytes in centralized clinical laboratories. Multiplexed PoC testing is a simultaneous on-site detection method for different analytes from a single specimen, especially at the point-of-care (PoC) settings, where an immediate decision on treatment needs to be made. Therefore, multiplexed detection by PoC system ensures the quality and performance of in vitro diagnostics, which will pave the way for novel health monitoring at home and add valuable information for personalized medicine. PoC testing decentralizes the traditional laboratory setting that greatly reduce the risk and increase interaction of laboratory personnel with patients and other healthcare team.
Our previous invention (PCT/TR2020/050115) relates to the immuno-optomagnetic Point-of-Care (PoC) assay and method for detection of hErbB2 (Her2) serum biomarker specific to breast cancer. This patent document describes a combination of following two major components for detection of a model biomarker (hErbB2 protein) in serum; (i) pre-activated PoC-chip platform that carried a Ab1 antibody on chip, and (ii) bioactivated magnetic quantum dots (MQDs-Ab2-x). The assay method originally designed to carry out on a PoC-chip platform in two sequential steps. First, MQDs-Ab2-x specifically captures protein biomarker analyte (hErbB2 protein) from the test serum samples to form “Complex-1”. Second, this Complex-1 is then sandwiched on pre-activated PoC chip to form a sandwich Complex-2. The Complex-2 can later be visualized for the detection and quantification of analyte.
The above process related to our previous invention was although sensitive, but it is relatively tedious and require more cost for the whole assay to perform. Therefore, in the present invention, the assay method/process is re-designed to provide more simpler and cost-effective approach to in vitro PoC assay method/process that eliminates the requirement of a PoC-chip component and reducing the cost of the assay process/method. Also, speed of assay and real-time detection of diagnostic signal which is much simpler than the previously demonstrated assay/process in PCT/TR2020/050115.
Until now, there is no standard tool to diagnose MCI or AD before it precedes. Therefore, it is critically important to develop innovative method for a low-cost, equipment-free, accurate and early diagnosis of MCI or AD. Current understanding in relation to shaping future diagnosis of AD suggest that early diagnosis of AD can only be possible in a minimally invasive way through probing the bodily responses to the disease, such as screening for AD-specific biomarkers released in the body fluids.
There are a few known possibilities where the fractions of these biomarkers (cleaved oligomers) can be found in cerebral spinal fluid (CSF) or blood serum, or plasma. These oligomer fractions are diffusible proteins derived from amyloid-β (Aβ) and tau-protein or phosphorylated tau-protein (P-tau) that indicates AD-pathology and serve as signals for AD. Such proteins can be targeted for early diagnosis of MCI, AD or dementia. The current challenge is to develop an affordable, sensitive, equipment-free, and simple PoC assay and method for early AD diagnosis at a patient bedside or at homes.
In the present invention, multi-colored CdSe/CdS/ZnS non-magnetic free/colloidal quantum dots (QDs) and magnetic quantum dots (MQDs) were “re-purposed” for developing a new equipment-free “solution phase PoC in vitro assay”.
Following are the main differences between the present invention and our previous PCT/TR2020/050115 patent document:
Therefore, in the present invention, the assay method/process is re-designed to provide more simpler, rapid and costeffective approach for PoC in vitro assay method/process that eliminates the requirement of a PoC-chip component. The re-designed PoC assay in the present invention can be performed directly in the reaction tube and the detection signals can be directly monitored in real-time in free solution. This is accomplished by replacing the PoC-glass chip platform in previous invention with free, colloidal, and non-magnetic QDs-Ab1-(x) in the present invention for direct signal detection in the reaction solution in real-time without any additional isolation/purification steps.
In this invention, It is aimed to develop a rapid and simple immuno-optomagnetic point-of-care (PoC) in vitro assay/process using multiple (“four”) model disease biomarkers, such as Aβ1-40, Aβ1-42, tau-protein, and BDNF that are the indicators of Alzheimer's disease. These biomarker proteins are pathogenic amyloid fibrils that accumulate inside the human body during the course of aging, damage the brain and give rise to cerebral amyloid angiopathy, neuronal dysfunction and cellular toxicity that constitute the recognizable features of dementia, mild cognitive impairment and Alzheimer's disease. The in vitro immuno-optomagnetic assay/method developed utilizes a combination of multimodal features of bioactivated non-magnetic, colloidal/free QD nanocrystals as well as magnetic quantum dots (MQDs) to generate analyte-specific real-time quantitative/semi-quantitatively measurable signals for visible detection and quantification of multiple biomarker levels in serum.
The multiplexed biomarker detection is carried out using a combination of contrasting colored non-magnetic, fee/colloidal QDs and MQDs in a small microtube with a few tens of microliters volume. The assay process/method involves the following steps;
The first (Ab1) or a second (Ab2) antibody in this invention is not limited to a single protein biomarker or disease but also possibly extended to a variety of biomolecules, receptors, pathogenic bacteria, virus, DNA/RNA, environmental contaminants, pesticides or drugs which are generally described as analyte specific reagents in the present invention.
The main difference in the present invention compared to our previous invention of (PCT/TR2020/050115) are the following:
The developed in vitro immuno-optomagnetic PoC assay method/process potentially create a new opportunity and approach to clinical diagnosis of chronic diseases and generate a new market in healthcare sector. The proposed invention provides an alternate to current practices for the diagnosis of Alzheimer's disease that are not only expensive but also time consuming and difficult for elderly patients to frequent hospital visits. The PoC assay method/process designed in this invention, therefore potentially revolutionizes the elderly care by enabling easy and early disease diagnosis and monitoring options to track risk markers in serum or body fluids of suspected elderly individuals. The developed assay is not limited to above four biomarkers but also applicable to a wide range of analytes including bacterial/viral pathogens, drugs, environmental contaminants, or DNA/RNA.
The re-designed PoC assay in the present invention can be performed directly in the reaction tube instead of using a chip and the diagnostic signals can be directly monitored in real-time. This is accomplished by replacing the PoC-glass chip platform in previous invention with non-magnetic QDs-Ab1-(x) in the present invention for direct signal detection in the reaction solution without having to follow any additional step.
The main components in the present invention are as follows;
The advantages, including novelty/innovative aspects of proposed invention as well as superiority to prior art is summarized as follows:
This invention will therefore provide a versatile tool for clinicians, physicians, or surgeons to rapidly and accurately identifying elderly population who may be at high risk. It is also possible that PoC based detection assay will facilitate monitoring the progression of MCI, AD or dementia. Multiplexed detection by the PoC tests potentially also ensures the quality, accuracy and performance of in vitro diagnostics, which will pave the way for novel health monitoring at home and add value to personalized medicine.
The present invention describes an in vitro point-of-care (PoC) immuno-optomagnetic assay method/process for rapid and sensitive detection of multiple protein biomarker/s for Alzheimer's disease in free solution. The developed method utilizes a combination of non-magnetic quantum dots (QDs) and magnetic quantum dots (MQDs) that captures serum analytes to display change in color that is directly visible to a naked eye within 10-20 min. The assay has the flexibility and multifunctionality toward detecting a whole range of other diseases or biomarkers, pathogenic bacteria, virus, nucleic acids (DNA/RNA), toxic chemicals, pesticides or drugs.
A method for detecting and quantifying of a target analyte in a test sample for central nervous system disease comprises following steps of:
The present invention goes a step forward and extends the capabilities of previous immuno-optomagnetic PoC assay by following advancements:
In the current invention, the multi-colored non-magnetic CdSe/CdS/ZnS quantum dots (QDs) and magnetic quantum dots (MQDs) were “re-purposed” to develop a new equipment-free “solution phase PoC in vitro assay”.
In this invention, we developed a rapid and simple point-of-care (PoC) in vitro assay/process using multiple (“four”) disease biomarkers (‘z’) as models, where ‘z’ is either of the following biomarker proteins; (1) Aβ1-40, (2) Aβ1-42, (3) tau-protein, and (4) BDNF that are the indicators of Alzheimer's disease. The developed point-of-care in vitro method/process potentially replaces current practices for diagnosis of Alzheimer's disease that are time-consuming tests (see Annex. 1), or rely on heavily centralized facilities, such as MRI at hospitals at the stage when the individual already had visible signs of Alzheimer's disease, dementia, or mild cognitive impairment (MCI). The PoC assay method/process designed in this invention has the potential to revolutionize elderly disease diagnosis by utilizing serum or body fluids to rapidly detect for disease risk biomarkers. The developed assay is not limited to above four biomarkers (z), but also applicable to a wide range of analytes including bacterial/viral pathogens, drugs, environmental contaminants, or DNA/RNA.
Single color green CdSe core nanocrystals with gradient CdS and ZnS shell-shell were synthesized using a previously described method [16]. First, Se—S solution was prepared in a glovebox by dissolving 0.316 g (4 mmol) selenium powder and 0.128 g (4 mmol) sulfur powder in 3 mL TOP and placed in a desiccator under vacuum until use. In a separate three-necked flask, 0.025 g (0.2 mmol) CdO, 0.87 g (4 mmol) zinc acetate, 2.75 mL oleic acid and 10 mL octadecene were added and the flask was heated to 150° C. for 1 h under vacuum for degassing.
This was followed by introducing argon gas into the flask from an inlet connected to a condenser, while vacuum tube connected at the outlet and the reaction temperature was raised to 310° C. After stabilizing the temperature to 310° C., 1.5 mL of above Se—S solution was quickly injected to the solution while maintaining the temperature to 304° C. and waited for 20 min for reaction to complete. This procedure was optimized to obtain a single color (green) nanocrystals that were subjected to purification.
Single colored red CdSe core with gradient CdS and ZnS shells of nanocrystals were synthesized as described previously [17]. Here, instead of mixing the Se and S solutions together, they were prepared separately in a glovebox. For this, selenium solution was prepared by dissolving 0.158 g (2 mmol) Se powder in 0.89 mL (0.74 g) trioctylphosphine (TOP), while the sulfur solution was prepared separately by dissolving 0.192 g 23 mmol sulfur powder in 0.45 mL (0.37 g) TOP. These solutions were placed in a desiccator under vacuum until use.
In a separate three-necked flask, 0.09 g (0.7 mmol) CdO, 0.27 g (1.5 mmol) zinc acetate, 3.6 mL oleic acid and 15 mL octadecene was poured and degassed by heating at 150° C. for 1 h under vacuum. The argon flow at the condenser inlet was turned on and raised the reaction temperature in flask to 300° C. Once the temperature was stabilized, 0.1 g (1.2 mmol) of Se solution was quickly injected to the reaction mixture using a syringe needle, after 90 seconds, 0.018 mL dodecanethiol was added gradually by slow injection and the reaction was allowed to continue for 15 min. Lastly, 0.29 g sulfur solution prepared previously was injected into the reaction flask and waited 15 min for reaction to complete and removed the flask for cooling the reaction mixture.
About 15 mL of the reaction mixture containing red CdSe/CdS/ZnS nanocrystals was diluted with equal volume of chloroform and precipitated by adding 45 mL acetone and centrifuged for 15 min at 4500 rpm. The supernatant was removed, and the pellet was re-dissolved in 2 mL chloroform and re-precipitated by adding 6 mL acetone, centrifuged for 5 min at 4500 rpm and the process was repeated at least 8 times. Finally, the precipitate was dried at 60° C. overnight and the dried powder yielded 73 mg of CdSe/CdS/ZnS nanocrystals that was subjected to ligand exchange as described above.
The entire reaction mixture containing green/red nanocrystals and impurities were divided into several 1 mL aliquots. Each aliquot was diluted by adding 10 mL chloroform and the nanocrystals were precipitated by mixing with 30 mL acetone non-solvent and centrifuged for 2 h at 4500 rpm. The supernatant was collected in a separate tube that contained residual nanocrystals for further precipitation, while the pellet was dissolved in 2 mL chloroform and precipitated again by adding 6 mL acetone and centrifuged for 2 h at the same speed. This process was repeated 10 times and the final precipitate obtained was dried at 60° C. The dried nanocrystals were subjected to ligand exchange process for surface functionality and water solubility as described below.
Single color green or red QD nanocrystals synthesized above were subjected to surface coating through ligand exchange process using a combination of methods described previously [18, 19]. Briefly, synthesized pure and dried core-shell-shell green and red QD nanocrystals (2.85 mg each) were separately dissolved in 1 mL chloroform. In a separate vial, 0.25 g of L-glycine was dissolved in 5 mL deionized water and mixed with 0.2 mL of carbon disulfide and vortexed.
At this stage, a milky solution of functionalized QD nanocrystal was obtained (Scheme 1) which was added with 1 mL (2.85 mg in chloroform) of green or red core-shell-shell QD nanocrystals and the mixture was stirred for 24 h on a magnetic stirrer at room temperature. During this period, phase transfer of the QDs occurred from organic phase to aqueous phase following coating with a thin layer of dithiocarbamate and a hydrophilic poly-glycine layer, making the QDs more stabilized and colloidal in aqueous solution. The transfer of QDs to aqueous phase was visibly observed under the UV light as a conformation of ligand exchange reaction.
The nanocrystals from aqueous phase were aspirated and purified by diluting with deionized water in 1:4 ratio and precipitated by adding 20 volumes of acetone followed by centrifugation at 10000 rpm for 30 min for green QDs and 5000 rpm for 5 min for red QDs, respectively. The precipitate was then re-dispersed in aqueous buffer (PBS, pH 7.4). The water-soluble core-shell-shell QDs were later stored at 4° C. until use.
2. Synthesis of Magnetic Fe3O4 Nanoparticles (MNPs)
Magnetic Fe3O4 nanoparticles (MNPs) were synthesized using a previously reported co-precipitation method [20] and were size-fractionated at varying relative centrifugal force (RCF, g). Briefly, 0.25 mL (Fe+2) of 2 M FeCl2 ·4H2O in HCl was mixed with 1 mL of 1.1 M FeCl3 ·6H2O (Fe+3) in deionized water for 15 min and co-precipitated under alkaline conditions. The salt solution was heated for 10 min at 80° C. and the co-precipitation of Fe+2 and Fe+3 was initiated by slow dropwise addition of 3 mL of 3 M ammonia solution. MNPs obtained were separated with the help of a magnet and the MNPs residue was washed with double deionized subjected to gravity (g) fractionation at varying RCFs.
Gravity fractionation of MNPs was carried out and isolated different nano-sizes by applying RCF from 500-14500×g for 5-10 min, which enabled fractionating different nano-sized MNPs. Larger sized MNPs fraction that sedimented at 500-14500×g within 5 min were removed and only those fractions that were sedimented at 16000×g in 5 min were collected whose size ranged between 5-10 nm. These bare MNPs were subjected to stabilization and images were taken using an ultra-high-resolution TEM (JEOL JEM-ARM200CFEG UHR-TEM equipped with STEM, Cs corrected STEM and EDS).
As-synthesized bare MNPs were suspended in freshly prepared 4.5 mL of 0.3 M ammonia solution and the MNPs were dispersed by sonication using a probe ultrosonicator for 1 h. The colloidal MNPs were washed with five exchanges of 1 mL of deionized water followed by 3 min magnetic separation. The washed bare MNPs were dispersed in 4.5 mL of 50 mM L-aspartic acid and mixed by stirring vigorously for 6 h until the solution pH=2-3. The reaction pH was raised to 10-11 by dropwise addition of 3 M ammonia solution and the excess non-adsorbed aspartic acid was removed in successive magnetic separation and washing five-times with 2 mL dilute ammonia solution (0.3 M). Finally, the aspartic acid-stabilized Fe3O4 magnetic nanoparticles (Asp-MNPs) were stored in same solution until further use for chemically coupling with QD nanocrystals.
Polymer stabilized green/red CdSe/CdS/ZnS core-shell-shell QD nanocrystals carrying free carboxyl functionality were used for synthesis of green/red MQDs. Green MQDs (gMQDs) or red MQDs (rMQDs) were synthesized using non-magnetic water-soluble green QDs (gQDs) and red QDs (rQDs) as follows. First, 200 μL (0.57 mg) of water-soluble green QDs were suspended in 500 μL deionized water and vortexed for 5 min. To this, 100 μL of freshly prepared 150 mM of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and incubated for 5 min followed by addition of 100 μL of 300 mM N-hydroxysuccinimide (NHS) and the reaction mixture was again incubated for 5 min under static condition. The resulting EDC-activated bright fluorescent nanocrystals were quickly mixed with 1 mL of 10 mg/mL of Asp-MNPs (5-10 nm) suspension in deionized water and incubated for 2 h for covalent coupling under vigorous shaking at room temperature. The resulting reaction mixture containing hybrid MNPs@CdSe/CdS/ZnS nanoparticles (magnetic quantum dots, MQDs) were magnetically separated and washed by resuspending in PBS solution, pH 7.4. Coating of MNPs on nanocrystal surfaces was confirmed visibly after magnetically separating gMQDs and rMQDs separately that appeared brightly illuminating magnetic nanoparticles upon exposure to UV irradiation at 365 nm, which accompanied by disappearance of fluorescence from the supernatant solution.
4. Bio-Functionalization of QDs with Polyclonal (Ab1) and MQDs with Monoclonal Antibodies (Ab2)
Red/green (r/g) QDs and MQDs conjugated with a specific antibody (Ab1 and Ab2) is termed as QDs-Ab1 and MQDs-Ab2, and the type of specific antibodies ‘x’ is assigned to any of the following antibody types:
The above four distinct antibodies are plurally termed as ‘x’ that represent either of the above three types of Abs for convenience. For example, MQDs-Ab2(x) represents MQDs conjugated with either of the above antibody types (a, b, c, or d), for instance rMQDs bioconjugated with anti-Aβ1-40 antibody is termed as rMQDs-Ab-a.
Bioconjugation was carried out using 20 mg of different colored (r/g) MQDs such as green (λ565, gMQDs) or red (λ655, rMQDs) MQDs. Similarly, bioconjugation of 4.32 mg different colored non-magnetic QDs was carried out, such as green-QDs (λ565, gQDs) or red-QDs (λ655, rQDs). Here, bioconjugation of antibodies specific to biomarker protein was designed to pair with distinct colored magnetic and non-magnetic QDs (MQDs:QDs). For instance, green MQDs (gMQDs) always paired with non-magnetic red QDs (rQDs) and vice versa.
Bioconjugation was carried out for non-magnetic QDs, for instance 4.32 mg of rQDs (λ655) or oQDs (λ585) or 20 mg of gMQDs (λ565) with free carboxyl-functionality were dispersed each in 100 μL of deionized water. To this, 100 μL mixture containing 50 mM EDC and 100 mM NHS in deionized water was mixed and incubated for 5 min. The surface activated gMQDs were magnetically separated, while surface activated non-magnetic rQDs were centrifuged at 14000 rpm for 5 min and the supernatant was discarded. The resulting nanocrystals were resuspended in 400 μL PBS solution (pH 7.4) and washed thrice by magnetic separation and centrifugation for gMQDs and rQDs, respectively. The activated gMQDs and rQDs (or rMQDs and gQDs) pellets were mixed each with 10 μL of 1 mg·mL−1 polyclonal and monoclonal antibodies, respectively and incubated at 4° C. for 5 h. After incubation, the g/rMQDs-Ab2-(x) bioconjugates (MQD-Ab2-a, MQD-Ab2-b, MQD-Ab2-c, and MQD-Ab2-d) were separated by magnetic separation and non-magnetic g/rQDs-Ab1(x) (QD-Ab1-a, QD-Ab1-b, QD-Ab1-c, and QD-Ab1-d) were separated by centrifugation at 14000 rpm for 5 min and respective supernatants were discarded. The above two processes were repeated thrice to wash bioconjugates (QDs-Ab1(x) and MQDs-Ab2-(x)) that were subjected to blocking step.
In addition, bare Asp-MNPs were conjugated with polyclonal antibodies (MNPs-x) as described above for MQDs-Ab2-(x). The free-functional groups on bioconjugates were blocked by adding 400 μL of 5% bovine serum albumin (BSA) in PBS solution (pH 7.4) in each reaction tube and incubated at 4° C. for 2 h. Finally, the bioconjugates were separated and washed with PBS in three cycles following magnetic separation or centrifugation, and finally resuspended in 400 μL of same buffer and stored at 4° C. until use.
The purified bioconjugates with any free active functional groups were blocked by incubating with 5% BSA in PBS solution (pH 7.4) containing 0.05% tween 20 (blocking buffer) for 30 min. For negative controls, non-magnetic QDs and MQDs were also incubated with only BSA to prepare QD-BSA or MQD-BSA protein conjugates under standard conditions. Finally, all bioconjugates were washed with PBS in three cycles following magnetic separation or centrifugation for magnetic and non-magnetic QD nanoparticles, respectively and resuspended in 400 μL of same buffer. All bioconjugates were again divided into several 10 μL aliquots and stored at 4° C. until use for in-vitro immunooptomagnetic detection assay.
Normal serum from human plasma (2.0 mL, male AB plasma, Sigma-Aldrich) was first treated by incubating with 10 mg of non-fluorescent MNPs-Ab1-(x) for 1 h to remove pre-existing or background target protein that may be present in the serum. The resulting MNPs-Ab1 were magnetically separated from target protein analyte-free serum. The above process was repeated at least thrice with same serum but by incubating with a 10 mg of fresh aliquot of MNPs-Ab1-(x).
Finally, the serum sample devoid of any analyte protein traces was used for the preparation of known levels of analyte protein (y) spiked serum samples. Here, the analyte protein (y) generally refers to any of the following analyte proteins; (i) Aβ1-40 (Invitrogen), (ii) Aβ1-42 (Abcam, Cambridge, MA), (iii) tau-protein (R&D Systems), and (iv) Brain Derived Neurotrophic Factor (BDNF, Fitzgerald). The pure analyte proteins (y) were first reconstituted as per the manufacturer's instructions and diluted with analyte-free serum to obtain a working stock of 20 ng mL−1 of specific target protein, and this sample was again serially diluted using analyte-free serum to obtain a series of concentration of analyte protein (0-20 ng mL−1). For negative controls, serum samples were spiked with BSA protein instead of target analyte protein under identical conditions as described above. These analyte protein/BSA spiked serum samples were utilized for in-vitro immuno-optomagnetic sensing assays.
In vitro screening for specific binding and specificity of MQDs-Ab2-(x) was carried out using antigen-free serum sample “spiked” with known concentrations of respective antigens (Aβ1-40, Aβ1-42, tau protein or BDNF). Here a series of varying antigen concentrations (0-20 ng/mL) for each type of antigen was incubated with constant amounts of nonmagnetic QDs-Ab1-(x) previously coated with ‘(x)’ antibodies. The antigen-antibody complex on non-magnetic QDs were then presented to specific antibody coated MQDs-Ab2-(x) as shown in Scheme 2(a-e) as an example for Aβ1-42 detection. The non-magnetic QDs and MQDs were of different color for probing color changes post-incubation of antigens. The unbound or free non-magnetic QDs or free antigen can be easily detected by measuring the residual fluorescence of non-magnetic QDs to calculate the bound antigen in the sample upon magnetic separation of MQDs-Ab2-(x).
Immuno-optomagnetic assay was carried out using a constant 15 μL (162 ng) of non-magnetic QDs-Ab1-(x) for each reaction tube in a series of spiked serum samples containing 0, 0.31, 0.62, 1.2, 2.5, 5 and 10 ng mL−1 of antigen protein in replicates (n=3) and incubated for 15 min at 30° C. This reaction was then followed by mixing constant 15 μL (2 mg) of MQDs-Ab2-(x) conjugates to each reaction tube and the volume was adjusted to a final 80 μL using antigen-free serum and mixed. Here the term ‘x’ in non-magentic QD-Ab1-(x) and MQDs-Ab2-(x) is plurally identifies any of the following specific antibodies; Aβ1-40, Aβ1-42, tau protein or BDNF proteins. For negative controls, (i) BSA protein was used instead of antigen protein under identical conditions or (ii) swapped with non-antigen proteins. The reaction tubes were then incubated again at 30° C. for 15 min and the detection signal was directly observed by exposing the reaction tube/s to the UV-LED (λ365) torch, and the changes in fluorescence colors due to the formation of bioconjugates-antigen protein complex were recorded before and after incubation. Here, the non-magentic QD-Ab1-(x) first captures target antigen molecules to give rise to a QD-Ab1-(x)+target primary complex (Complex-1) in serum which is later presented to sandwich by immuno-reaction with green MQDs-Ab2-(x) conjugates. The resulting secondary complex (Complex-2) yields a tertiary color for instance, faint orange colored fluorescence to the reaction mixture from its original red and green. The resulting Complex-2 of MQDs-Ab2-(x)+target+non-magnetic-QD-Ab1-(x) formed is magnetically separated and a distinctive tertiary colored fluorescence from parent colors is measured or directly visualized using a naked eye in real-time (Scheme 2(a-g)). The residual fluorescence from the supernatant containing free non-magnetic QD-Ab1 is measured using a spectrofluorometer (Nanodrop 3300). This residual fluorescence provided an accurate measurement of the concentration of target biomarker/s (Aβ1-40, Aβ1-42, tau protein or BDNF proteins) captured by MQDs-Ab2-(a-d) in the reaction mixture. The fluorescence intensity of bound Complex-1 was calculated using the measured residual fluorescence intensity.
Specificity of the immuno-optomagnetic assay was tested under identical conditions as described above, except that in place of the target protein, non-specific proteins, such as BSA or antigen swapping (e.g., non-specific and truncated antigen namely AP-20) was utilized.
In this invention, we aimed at developing in vitro PoC assay method/process using model multiple (“four”) disease biomarkers, such as Aβ1-40, Aβ1-42, tau-protein, and BDNF that particularly represent for an Alzheimer's disease. The PoC assay/process designed is not limited to above four biomarkers but can also be plurally applicable to sensing various other disease biomarkers, pathogens, drugs, environmental contaminants, or DNA/RNA. The model biomarker proteins targets used in this invention, such as Aβ1-40, Aβ1-42 and related various amino acid length fragments are variants of Aβ proteins that are regarded as pathogenic amyloid fibrils.
These fibrils are found to accumulate inside the human body during the course of aging, damage brain and cause cerebral amyloid angiopathy, neuronal dysfunction as well as cellular toxicity that are characteristics features of an Alzheimer's disease [21, 22]. Tau protein and BDNF also have crucial roles in the pathogenesis of Alzheimer's disease [23, 24].
For better clarity following terminologies have been adapted in this invention:
Red/green (r/g) freely suspended, colloidal and non-magnetic QDs were bioconjugated with specific antibodies are designated as QDs-Ab1. The magnetic quantum dots (MQDs) bioconjugated with a specific antibody is designated as MQDs-Ab2. The type of antibodies plurally/generally designated (termed) as ‘x’ that represent any one of the following antibody types depending upon specific assay carried out related to it:
For example, MQDs-Ab2(x) represents MQDs conjugated with either of the above antibody types (a, b, c, or d), for instance rMQDs bioconjugated with anti-Aβ1-40 antibody is termed as rMQDs-Ab-a.
Similarly, serum biomarkers or analytes plurally designated as ‘z’, where z is either of the following four biomarker proteins that are representative indicators of Alzheimer's disease:
Here, QDs-Ab1-(x) and MQDs-Ab2-(x) bind to specific analyte ‘z’, for example:
Immuno-optomagnetic assay was carried out using a constant 15 μL (162 ng) of non-magnetic QDs-Ab1-(x) for each reaction tube in a series of spiked serum samples containing 0, 0.31, 0.62, 1.2, 2.5, 5 and 10 ng mL−1 of antigen protein ‘z’ in replicates (n=3) and incubated for 15 min at 30° C. This reaction was then followed by mixing constant 15 μL (2 mg) of MQDs-Ab2-(x) conjugates to each reaction tube and the volume was adjusted to a final 80 μL using antigen-free serum and mixed. Here the term ‘x’ in non-magentic QD-Ab1-(x) and MQDs-Ab2-(x) plurally identifies any of the following specific antibodies specific to analyte proteins ‘z’, where z=(1) Aβ1-40, (2) Aβ1-42, (3) tau protein or (4) BDNF proteins. For negative controls, (i) BSA protein was used instead of antigen protein under identical conditions or (ii) swapped with non-antigen proteins, for instance analogue Aβ1-20 protein. The reaction tubes were then incubated again at 30° C. for 15 min and the detection signal was directly observed by exposing the reaction tube/s to the UV-LED (λ365) torch, and the changes in fluorescence colors due to the formation of bioconjugates-antigen protein complex were recorded before and after incubation. Here, the non-magentic QD-Ab1-(x) first captures target antigen molecules to give rise to a QD-Ab1-(x)+target primary complex (Complex-1) in serum which is later presented to sandwich by immuno-reaction with green MQDs-Ab2-(x) conjugates. The resulting secondary complex (Complex-2) yields a tertiary color for instance, faint orange colored fluorescence to the reaction mixture from its original red and green. The resulting Complex-2 of MQDs-Ab2-(x)+target+non-magnetic-QD-Ab1-(x) formed is magnetically separated and a distinctive tertiary colored fluorescence from parent colors is measured or directly visualized using a naked eye in real-time (Scheme 2(a-g)). The residual fluorescence from the supernatant containing free non-magnetic QD-Ab1-(x) is measured using a spectrofluorometer (Nanodrop 3300). This residual fluorescence provided an accurate measurement of the concentration of target biomarker/s (‘z’) captured by Complex-2 containing QDs-Ab1-(x) and MQDs-Ab2-(x) in the reaction mixture (QDs-Ab1-(x)-z-(x)-Ab2-MQDs). The fluorescence intensity of bound Complex-1 was calculated using the measured residual fluorescence intensity, while the change in the fluorescence color is directly visualized by a naked eye in real-time.
11.1. PoC In Vitro Binding and Specificity Test Using rQDs-Ab1-a gMQDs-Ab2-a and Serum Analyte Aβ1-40 Protein
To a series of spiked serum samples containing varying concentrations of known Aβ1-40 protein from 0 to 20 ng mL−1 was incubated each with a constant 162 ng (15 μL) of non-magnetic QDs-Ab1-(x) and incubated for 15 min at 30° C. to form a Complex-1 as described in experimental methods. Each of these tubes were then added with a constant 2 mg (15 μL) of MQDs-Ab2-a to capture and sandwich with Complex-1 that gave rise to a Complex-2. This Complex-2 provided a distinctive fluorescent color which is different from the parent colors that was proportional to the analyte concentration (Aβ1-40 protein), which provided a semi-quantitative (visible to naked eye) and it was quantitatively estimated to determine the analyte Aβ1-40 protein concentration present in the test serum.
11.2. PoC in vitro binding and specificity test using gMQDs-Ab2-b, rQDs-Ab1-b and analyte Aβ1-42
In this invention, the designed assay/method for detecting Aβ1-40 was applied to another pathogenic variant of amyloid β (Aβ) protein biomarker, namely Aβ1-42 whose molecular structure differs by the presence of two additional amino-acids in Aβ1-42 compared to Aβ1-40. Both Aβ1-40 and Aβ1-42 are pathogenic amyloid fibrils that accumulate inside the human body during the course of aging, damage the brain and give rise to cerebral amyloid angiopathy, neuronal dysfunction and cellular toxicity that are associated with the Alzheimer's disease [21, 22]. Results of immuno-optomagnetic in vitro assay for sensing Aβ1-42 is shown in
11.3. In vitro binding and specificity assay using MQDs-Ab2-c and QDs-Ab1-c with structurally distinct proteins but represents for same disease (Alzheimer's disease)
Further extension of the developed immuno-optomagnetic assay method/process was applied to two distinct biomarker proteins, such as tau-protein and BDNF proteins that also represent as biomarkers of Alzheimer's disease.
Detecting a single biomarker for a particular disease presents potential risk of inaccuracy or false positive or false negative results. Therefore, detecting a panel of multiple biomarkers for a single disease for diagnosis maximizes the overall diagnostic accuracy in medical diagnosis. Therefore, in this invention, we extended the developed immunooptomagnetic in vitro assay method/process to other AD biomarkers, such as tau-protein and BDNF to enhance the assay performance in its accuracy for diagnosing the Alzheimer's disease.
11.4. In vitro binding and specificity assay using MQDs-Ab2-c and tau protein:
In this experiment, to distinguish from other experiments, the color of MQDs-Ab2-c and non-magnetic QDs were swapped. Instead of green MQDs, here we utilized red MQDs for distinctions from above two biomarkers.
Sensitive and specific capturing of antigen is clearly visible from spectra until saturation at 1.25 ng·mL−1 tau protein which can be clearly observed form the relative fluorescence intensity values at λ565 and the data were fitted with a non-linear regression equation that showed binding curve saturation in
11.5. In vitro binding and specificity assay using MQDs-Ab2-d, QDs-Ab1-c and BDNF protein:
Multiplexed detection by the developed in vitro PoC immuno-optomagnetic assay potentially ensures the quality, accuracy, and performance of in vitro diagnostics, which will pave the way for novel health monitoring at homes and add value to personalized medicine. Finally, the success and outcomes from this invention will address the current challenges in the field of clinical diagnosis, allow continuously monitoring the disease in the elderly patients, help improving elderly/aged health, provide opportunity for the early detection of disease and prevention, enhance healthy lifespan, compete with existing expensive biomedical practices and opportunity to enter a new global in vitro diagnostic market and improve overall socio-economic development.
This list of references cited by the applicant is for the reader's convenience only. It does not form part of the patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded in this regard.
Non-patent literature cited in the description
This application is the national phase entry of International Application No. PCT/TR2021/051627, filed on Dec. 30, 2021, the entire contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/TR2021/051627 | 12/30/2021 | WO |
