Developing a Molecular 'Trojan Horse' for the Earlier, Minimally-Invasive Diagnosis of Alzheimer's Disease

Alzheimer’s is a neurodegenerative disease that gets progressively worse with age so an early diagnosis is essential to ensure medication is most effective. Current diagnostic tools identify amyloid beta (Aβ) plaques, which are present during the later stages of the disease. I realised there was a necessity for an earlier diagnostic tool and decided to investigate further.

Aβ oligomers are present in much higher concentrations in the brains of Alzheimer’s patients and this increase appears during the earliest stage of the disease, making Aβ oligomers an attractive biomarker. This led me to develop a diagnostic probe that could effectively target Aβ oligomers. I engineered a molecular 'Trojan Horse' that had the potential to do this and cross the blood-brain barrier, a major obstacle for current Alzheimer's diagnostic probes. I determined the probes' optical properties and performed surface plasmon resonance to estimate their affinity to Aβ oligomers and to transferrin receptors (TfRs). The probes' cross-reactivity with various other species of Aβ was also determined.              

The probes exhibited optical properties that were suitable for non-invasive imaging. The probes also successfully targeted Aβ oligomers and TfRs but their affinity to TfRs potentially allows them to cross the blood-brain barrier. These molecular 'Trojan Horses' have the potential to cross the blood-brain barrier and be used as a sensitive, minimally-invasive tool for the earlier diagnosis of Alzheimer's Disease! This same concept could also potentially be used for the minimally-invasive diagnosis of other amyloidogenic neurodegenerative diseases such as Parkinson's and could help millions of people!  





Hi, my name is Krtin Nithiyanandam and I live in Surrey, England. I am 14 years old and a Year 10 student at Sutton Grammar School. I love playing squash, playing the piano and science. I actually first became interested in science, specifically the medical sciences, after my second operation. I was fascinated by how specific drugs worked inside our bodies and how scientists are developing new therapuetics to make everyday life better. 

My scientific idol is Albert Einstein, not because he was a brilliant scientist but because he was a teacher of moral human values, he wanted to inspire people to ask "Why?" My favourite quotation from him is, "The important thing is to never stop questioning." 

When I am older, I would love to study medicine as this would allow me to learn about current drugs and help people at the same time. I would also like to become a medical researcher at some point in my life as I want to help develop new drugs and discover new things that could benefit mankind.  I was also greatly inspired to pursue a career in medicine and science by my Year 9 biology teacher, Mr Storey. 

Winning the Google Science Fair would be incredible and truly life-changing as it would encourage me in my pursuit of studying medicine and it would also allow me to meet like-minded competitors and research professionals who share the same interests in science that I do!



Current diagnostic tools identify amyloid-beta (Aß) plaques, which are present during the later stages of Alzheimer's disease. Newer research has also suggested that Aß plaques are present in healthy individuals, making them an ineffective biomarker for the disease. Aß oligomers are present during the earliest stages of the disease and are in significantly higher concentrations in the brains of Alzheimer's patients in comparison to healthy individuals, making them an attractive biomarker.        

Question: Given the necessity for an earlier diagnostic tool, can I develop a novel, minimally-invasive diagnostic probe that could be used for the earlier diagnosis of Alzheimer's by targeting Aß oligomers and potentially crossing the blood-brain barrier(BBB) using receptor-mediated transcytosis?    

Hypothesis: I hypothesised that a bispecific antibody(BsAb), engineered from a an anti-TfR antibody (IgM) Fab' and an anti-Aß oligomer-specific antibody (IgG1) Fab', could be used as an effective diagnostic probe. I hypothesised that the BsAb would have a low affinity to TfRs(as IgMs tend to have low affinity), thus potentially allowing it cross the BBB using receptor-mediated transcytosis. I also hypothesised that due to the IgG1 Fab' of the anti-Aß oligomer-specific antibody in the BsAb, the BsAb would have a high affinity to and successfully target Aß oligomers but not Aß plaques or monomers, making the BsAb more sensitive. I hypothesised that by conjugating the BsAb to quantum dots(QDs), these probes could be used for a minimally-invasive diagnosis as QDs can emit light in the Near Infrared Region(NIR), thus allowing the probes to be detected non-invasively using fNIR-Spectroscopy.              



Alzheimer's disease is a neurodegenerative disorder that gets progressively worse and an early diagnosis is essential to ensure treatment is more effective. 

Identifying the appropriate biomarker:   

Current diagnostic tools identify beta-amyloid(Aβ) plaques - which are present during the later stages of the disease. New research has also suggested that Aβ plaques and monomers are present at similar concentrations in the brains of Alzheimer's patients and healthy individuals, making them ineffective biomarkers for the disease. Aβ oligomers are present during the earliest stages of the disease and are in significantly higher concentrations in the brains of Alzheimer's patients, making them attractive biomarkers. Aβ1-42 is the predominant form of Aβ found in the brains of Alzheimer's patients and so my experiments were conducted using Aβ1-42.   

fNIR Spectroscopy

Visible light has poor transmission through biological tissue however light in the Near Infrared Region (NIR) can be used for deep tissue imaging as there is increased penetration and limited absorption and scattering of NIR light by biological tissue. The NIR for in vivo imaging is between 700nm - 900nm and fluorophores that emit light in the NIR can be detected non-invasively using fNIR Spectroscopy.     

Quantum Dots


Quantum dots (QDs) have unique properties and structure (Figure (1)) that make them superior to organic fluorophores - QDs have higher fluorescence and improved photostability. The diameter of the QDs are smaller than that of the Bohr radius (0.0529177nm), therefore the excitons are squeezed, leading to the quantum confinement effect (Figure (2)). The larger the QD, the less energy is required to confine the exciton and photons are released with less energy. Smaller QDs emit UV light whilst larger QDs emit IR light. QDs can also emit light in the NIR, making them suitable for fNIR Spectroscopy. 

Crossing the blood-brain barrier- Receptor-mediated transcytosis

The BBB is a highly selective permeable barrier formed by capillary endothelial cells. This ensures that very few objects can reach the brain, advantageous for protecting your brain from invading pathogens but detrimental as very few therapeutics can reach the brain. Researchers have discovered that antibodies with a low affinity to TfRs can cross the BBB via receptor mediated transcytosis. The anti-TfR antibody binds to TfRs and is transported across the endothelial cell, but due to the low affinity of the antibody, when it reaches the other side of the endothelial cell, it is released from the TfRs and into the brain.        

Existing Research: 

Little research was present on using QDs for a targeted diagnosis of Alzheimer's. Tokuraku et al. investigated into developing a QD probe but these probes were not specific to Aβ and only had applications for in vitro studies of Aβ aggregation. Feng et al. tried to develop a QD probe specific to Aβ however the probe could not cross the BBB and targeted all forms of Aβ - extremely detrimental for diagnosis.   

Using this research, I developed a QD probe that had the potential to cross the BBB, be detected non-invasively using fNIR Spectroscopy and was specific to Aβ oligomers.    


(mAB) Anti-Aß oligomer-specific antibody (IgG1) F(ab')2 generation: 0.5mL of antibody(8mg/mL) was added to equilibriated immobilised ficin column and incubated(37ºC) for 25hours. Generated F(ab')2 fragments were purified with NAb Protein A Column and centrifuged(1000xg) for 1minute. Flow-through concentration was determined spectrophotometrically by measuring absorbance at 280nm.        

(mAB) Anti-TfR antibody (IgM) F(ab')2 generation: Equilibriated immobilised pepsin column was washed with 8mL IgM F(ab')2 digestion buffer(200ml, 100mM sodium acetate, 150mM NaCl, 0.05%NaN3; pH4.5). Column and 1mL of antibody(1mg/mL) were incubated(37ºC) separately for 3 minutes. Antibody was added to column and incubated(37ºC) for 1.5hours. Generated F(ab')2 fragments were centrifuged in C30 Concentrator and concentration was determined spectrophotmetrically by measuring absorbance at 595nm.             

Fab' Generation: 1mL of anti-Aß oligomer-specific antibody F(ab')2(10mg/mL) was dissolved in 20mM buffer(sodium phosphate, 0.15M NaCl, 5mM EDTA,pH7.4). 6mg of 2-MEAHCl was added and incubated(37ºC) for 1.5hours. Excess 2-MEAHCl was removed by gel-filtration. Protocol was repeated for anti-TfR antibody Fab' generation.

BsAb synthesis: Anti-Aß oligomer-specific antibody Fab' (Fab'A) was added to DTNB(40mg DTNB, 10ml 1MTris‐HCl, pH7.5) and incubated(RT). Equimolar ratios of Fab'A-DTNB and anti-TfR antibody(Fab'B) were mixed and incubated(37ºC) for 1.5hours. Reaction was incubated(4ºC ) overnight. BsAb fraction was purified with Superdex 200 column equilibriated in PBS. Each stage was monitored by HPLC.


BsAb and QD(Qdot800(CdSe/ZnS)) conjugation: 25mM QDot800(100µg/mg) and BsAb(1mg/mL) solutions were prepared separately in PIPES buffer. 20mM EDC, 50mM sulfo-NHS solution was prepared immediately before use. 250µL EDC/sulfo-NHS was added to QD solution. Reaction was incubated(RT) for 10minutes and 7µL of 2-MEA was added to quench EDC. 25µL of BsAb solution was added to activated QDs. Reaction was incubated(RT) for 60minutes. Excess reactants and sulfo-NHS were removed by dialysis against 50mM Tris, pH7.4.


 1-42 oligomerisation: Aß1-42 peptide was dissolved in 100% HFIP(100mL, 1mg/mL), incubated(RT) for 1hour and then sonicated. Solution was lyophilised under a gentle stream of nitrogen gas and resuspended in 100% DMSO. Solution was incubated(RT) for 20minutes, aliquoted and stored at -80ºC. When required, 500μl D-PBS was added and incubated for 2hours-4hours(dependent on required oligomer 'size'). Aß oligomer molecular-weight was determined by gel electrophoresis.

Absorption spectra and emission spectraAbsorption(excitation) spectra(450nm-850nm) and emission spectra(700nm-900nm) of 1mM of QDot800(CdSe/ZnS), AlexaFluor790 and QD-probes in PBS were recorded using SpectraMax Plus 384 Microplate Reader. Absorption was plotted in terms of extinction coefficients.                   

QD-probe affinity: Surface plasmon resonance(Biacore) was used to determine affinity. 0.4M EDC/1M NHS solution was added to dextran matrix at flow rate of 10µl/min for 7min to activate surface. TfR solution(ligand, 50µg/mL, PBS diluent) was added at flow rate of 10µl/min for 7min. 1M ethanolamine-HCl(pH8.5) was added at flow rate of 10µl/min for 7min to deactivate excess reactive groups. Various concentrations of QD-probe solutions(analyte, PBS diluent) including duplicate-concentrations were used. Unmodified surface was used for reference analysis. Protocol was repeated using Aß oligomers of various sizes as ligand.          

Direct-fluorescence assay:  Aß monomers, fibrils, plaques and oligomers(100pg/ml-800pg/ml)  were blocked in PBS(w/ 5% BSA) in 384 well plates. The probes were added and incubated(RT) for 1hour, then washed with PBS-T. Fluorescence was read with a plate reader at 800nm(488nm excitation).                  








My results show that QDot800(QD(CdSe/ZnS)) and AlexaFluor790(organic dye(OD)) emitted light maximally at 800nm and 805nm respectively, making them both suitable for non-invasive fNIR-Spectroscopy.  

QDot800 had a 57x greater extinction coefficient(ε) than AlexaFluor790(at absorption maxima) and previous research(see References) has identified that QDs have fluorescence quantum yields(Φ) of 40%-90%(RT), which is higher than most ODs that emit light in the NIR, ultimately making QDs significantly brighter. Previous research has well established that QDs are significantly less susceptible to photobleaching due to their inorganic composition.        


Note: Absorption was plotted in terms of ε(10-6cm-1M-1) and emission intensity was plotted as relative %  

(Graphs have different scales for ε; ε is 57x greater for QDot800 than AlexaFluor790(absorption maxima))

ODs have narrow absorption spectra whereas QDs have broad absorption spectra, making QDs more practical as a single excitation source is required. ODs have broader emission peaks with significant emission intensity at wavelengths >850nm whereas QDs have narrow/asymmetrical emission peaks, enabling easier detection of QDs due to reduced inter-channel cross-talk.  

QDs have a large 'Stokes Shift' in comparison to ODs, allowing the detection of fluorescence whilst reducing interference. The QDs' large 'Stokes Shift' also reduces fluorescence quenching, resulting in a stronger signal.   

These results suggest that QDs are more suitable for non-invasive fNIR-Spectroscopy in comparison to ODs.             

 Overlay plot of sensorgrams from QD-probe steady-state affinity analysis was created. Sensorgrams were reference subtracted and blank subtracted. Response versus concentration plot was created and affinity(Kd) was read.  



 Kd was read as 1.54x10-4 moles. This high Kd value suggests that the QD-probe has low affinity to TfRs but can still engage TfRs, suggesting that it can cross the BBB via receptor-mediated transcytosis. This low affinity to TfRs can be expected due to the(IgM) anti-TfR antibody Fab' in the BsAb as IgMs tend to have low affinities.           

Aß oligomers

 QD-probes had loKd values(low-nanomolar(10-9) to high-picomolar(10-10) range) to Aß oligomers of varying molecular-weight, suggesting the QD-probe has a high affinity to Aß oligomers. This high affinity to TfRs can be expected due to the(IgG1) anti-Aß oligomer-specific antibody Fab' in the BsAb as IgG1s tend to have high affinities.      

QD-probe has a Kd value to TfRs that suggests it can cross the BBB and a Kd value that suggests it has a high affinity to Aß oligomers - demonstrating the QD-probe's potential as a diagnostic tool for Alzheimer's.     

QD-probe lost the asymmetrical emission peak however in comparison to ODs, they had a narrow emission spectra with maximum emission intensity at 800nm, making them suitable for non-invasive fNIR-Spectroscopy. 

QD-probe had 74% emission intensity compared to unconjugated-QDs however due to the high ε and Φ of QDs, the QD-probe would be significantly brighter than an OD-probe.     

Significant fluorescence was emitted from targeted Aß-oligomers and insignificant fluorescence was detected from other Aß species. 


QD-probe favoured Aß-oligomeric species and had limited cross-reactivity with other species of Aß, reducing chances of misdiagnosis. Fluorescence emitted from QD-probe was dependent on Aß-oligomer concentration and size, suggesting this probe can also determine the stage of the disease.  


Alzheimer's affects over 47 million people worldwide with 8 million new cases very year. The WHO identifies early diagnosis as essential to treat Alzheimer's however patients are usually diagnosed during the middle or later stages of the disease - after irreversible damage has taken place. Some patients suffering from Alzheimer's do not receive a diagnosis and this disease is estimated to cost US$604 billion to society by 2020. The  molecular 'Trojan Horse' I have developed could potentially be used for the earlier, minimally-invasive diagnosis of Alzheimer's Disease.     

By conjugating a bispecific antibody (BsAb) to selective wavelength quantum dots (QDs), I have created a diagnostic probe with promising affinities which can be non-invasively detected. The probe had a low affinity to transferrin receptors (TfRs), thus potentially allowing it to cross the blood brain barrier (BBB) via receptor mediated transytosis. It also had a promisingly high affinity to amyloid-beta (Aß) oligomers - which is beneficial for diagnosis. The CdSe/ZnS QDs used could emit light maximally at 800nm (NIR), thus allowing these probes to be detected non-invasively using fNIR-Spectroscopy. The QDs were significantly brighter and had various advantages over conventional organic fluorophores. The probes successfully targeted Aß oligomers also had very little cross-reactivity with other species of Aß. These results supported my hypothesis as the probes had non-invasive detection properties and had low affinities to TfRs - potentially allowing them to cross the BBB via receptor mediated transcytosis.   

 This biomarker is in significantly higher concentrations in the brains of Alzheimer's patients and the probe had little cross-reactivity with other species of Aß, reducing chances of misdiagnosis. Research has shown that Aß oligomers are the most neurotoxic form of Aß and are a major culprit of Alzheimer's Disease, and by targeting these oligomers, this probe could be used to diagnose the disease before all the damage has occurred - well before typical Alzheimer's symptoms surface. My results also suggest that this probe could be used to determine the stage of the disease by emitting levels of fluorescence dependent on Aß oligomer size and concentration. Due to the potential non-invasive detection of the QDs via fNIR-Spectroscopy and the probes' ability to cross the BBB, there would be no requirement for a lumbar puncture, making diagnosis minimally invasive.   

These findings have the potential to benefit millions of people worldwide and so I will aim to collaborate with various research institutes and organisations to help develop this diagnostic probe.

Future Work: I would like to study the probes in an in vitro BBB and potentially do some toxicology studies. I would also like to try and modify these probes so that they can diagnose other amyloidogenic neurodegenerative diseases by identifying different biomarkers.  

These molecular 'Trojan Horses' can be used for non-invasive imaging  and show significant promise for the earlier, minimally-invasive diagnosis of Alzheimer's Disease - with the potential to diagnose the disease before typical symptoms are present - allowing healthcare professional to administer treatment at earlier stages and ensure that the disease has fewer detrimental effects!  




Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target.

  • Yu YJ, Zhang Y, Kenrick M, Hoyte K, Luk W, Lu Y, Atwal J, Elliott JM, Prabhu S, Watts RJ, Dennis MS. (2011 May 25)

Bioconjugation Protocols and Strategies

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Bioconjugation Techniques

  • Greg T Hermanson (2008)

Cys diabody Quantum Dot Conjugates (ImmunoQdots) for Cancer Marker Detection

  • Bhaswati Barat,† Shannon Sirk,† Katelyn McCabe,† Jianqing Li,‡ Eric J Lepin,† Roland Remenyi, Ai Leen Koh,¶ Tove Olafsen,† Sanjiv S. Gambhir,§ Shimon Weiss,‡ and Anna M. Wu†*

Alzheimer's disease: β-amyloid plaque formation in human brain.

  • Seeman P, Seeman N.

Mechanism of amyloid plaque formation suggests an intracellular basis of Aβ pathogenicity

  • Ralf, Friedricha, Katharina Tepper,b,1, Raik Rönickec, Malle Sooma, Martin Westermannd,Klaus Reymannc, Christoph Kaethera, and Marcus Fändricha,b,e,2

Fluorescence imaging of APP in Alzheimer's disease with quantum dot or Cy3: a comparative study.

  • Feng L1, Li S, Xiao B, Chen S, Liu R, Zhang Y. (2010 25th March)

A quantum dot probe conjugated with aβ antibody for molecular imaging of Alzheimer's disease in a mouse model.

  • Feng L1, Long HY, Liu RK, Sun DN, Liu C, Long LL, Li Y, Chen S, Xiao B.(2013 May 22nd)

Structural conversion of neurotoxic amyloid-β1–42 oligomers to fibrils

  • Mahiuddin Ahmed, Judianne Davis, Darryl Aucoin, Takeshi Sato, Shivani Ahuja, Saburo Aimoto, James I Elliott, William E Van Nostrand & Steven O Smith (2010 March 5th)

Amyloid oligomers: formation and toxicity of Ab oligomers

  • Masafumi Sakono1,2 and Tamotsu Zako1(6th January 2010)

Amyloid-beta oligomerization in Alzheimer dementia versus high-pathology controls

  • Thomas J. Esparza BS1, Hanzhi Zhao BS1, John R. Cirrito PhD1,2,3, Nigel J. Cairns PhD1,3,4, Randall J. Bateman MD1,2,3, David M. Holtzman MD1,2,3,5 andDavid L. Brody MD, PhD1,2,* (December 7th 2012)


AlexaFluor Quantum Yields:


Quantum Dots for Live Cell and In Vivo Imaging

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Quantum dot bioconjugates for in vitro diagnostics & in vivo imaging

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I would like to thank Dr. A. Nithiraj for allowing me access to his lab and the School of Biosciences and Medicine, Department of Biochemistry and Physiology, for lending me resources, equipment and time. I would also like to thank him for teaching me how to operate Biacore and how to perform HPLC - however all experimental procedures were carried out by me and results were collected and analysed by me.  

A major source of support and help was from my school and I would like to thank my STEM teacher and my former chemistry teacher, Dr. Bass and Dr. Vines -  both of whom have PhDs and previous experience in the field of biochemistry,  for lending me old textbooks on various biochemical analytical techniques and for helping me understand the basics of fluorescence, excitation and emission at school before I carried out my project - even though it is not part of my GCSE curriculum. I would also like to thank my head of science, Mr Costello, for helping me enter and develop this project for this competition. I would also like to thank my former biology teacher, Mr Storey, who unfortunately left my school however we still remain in contact, for inspiring me to take an interest in biology and biochemistry. I would also like to thank my headmaster, Mr Ironside, and all my school-friends for supporting me during the course of this project. 

Finally, I would like to thank my parents for their invaluable support and for the time and effort they put in helping me get to places and for checking over the grammatical aspects of my work. 

Laboratory Health and Safety Documentation

(No carcinogenic compounds or severely hazardous chemicals were used in my project but the necessary safety precautions required if I were to use hazardous and carcinogenic compounds were covered in the Health and Safety form. Methanol as opposed to acetonitrile was used for HPLC).