Running

head: SELECTIVE 5-HT DETECTION 1

Selective 5-HT Detection: How Much of the Happy Drug is

in Your Blood?

(In partial fulfillment of requirements for the Honors Diploma)

Anna Stitt

Project Mentor: Dr. Jason Taylor

Roberts Wesleyan College

April 2018
Selective 5-HT Detection 2

Selective 5-HT Detection: How Much of the Happy Drug is in Your Blood?

1. Introduction

The fatal consequences and limited treatment options of Alzheimer’s disease plague

society today in a way unlike any other health concern. It is the most common form of senile

dementia in seniors and is characterized by neural apoptosis, deterioration of cognitive functions,

and the disruption of synaptic functions in the brain responsible for learning and forming

memories.1 The personal tragedy of this disease simply cannot be quantified by statistics;

imagine the glassy-eyed emptiness in a gaze from your loved one with absolutely no recognition.

Consider the day-in and day-out struggle of being a caregiver to your parent and losing the

relationship of mother and daughter. This emotional torment is not a rarity; according to The

Alzheimer’s Association, Alzheimer’s disease (AD) is the 6th leading cause of death in the

United States, accounting for one in every three senior deaths.2 More than 5 million Americans

live with the disease, and one more individual in the United States develops it every 66 seconds.2

This disease inflicts not only an emotional burden on the patient and their family, but a financial

burden as well. In 2017, Alzheimer’s and other forms of dementia cost the nation $259 billion; it

is estimated that this statistic will increase to $1.1 trillion in 2050.2 What makes AD most

despised is that there is neither a certain way to avoid its onset nor a method to overcome it. In

fact, while deaths from heart disease have decreased by 14% since 2000, the number of deaths

from Alzheimer’s disease has increased by 89%.2 Unfortunately, Alzheimer’s is only one disease

associated with a deficit of the neurotransmitter serotonin, or 5-hydroxy tryptamine (5-HT),

pictured structurally in Figure 1.

Selective 5-HT Detection 3

Figure 1: Structure of 5-Hydroxy Tryptamine (5-HT)

Before discussing the neurotransmitter serotonin, it is imperative to first develop an

understanding of neurotransmission and its importance in the body. Neurotransmission is

communication between neurons in the brain, which are nerve cells responsible for processing

and transmitting information across varying parts of the brain and to the rest of the nervous

system.3 They complete their job using chemical and electrical signaling because they have a

special ability to fire electrochemical pulses known as action potential.3 Dendrites, the branched

extensions of the nerve cells, play a key role in determining the extent to which action potentials

are produced by the nerve cells.3 Action potentials sent from one neuron travel rapidly down the

long neuron projection, or axon, causing other neurons to fire in response.3 When the action

potential occurs, neurotransmitters in one neuron are released into the synapse, or space between

two neurons.3 From here, the neurotransmitters, which are stored in synaptic vesicles inside of

neurons, can attach to receptors on another neuron.3 In this way, neurotransmitters chemically

link the brain and spinal cord to the rest of our bodies.

If neurotransmission processes are performed incorrectly, physiological and behavioral

abnormalities result, including Parkinson’s disease, Alzheimer’s disease, drug addictions,

depression, and insomnia.3 Serotonin, in particular, is imperative for its control of many

metabolic functions, such as mood, appetite, and sleep.3 Symptoms associated with a deficit of
Selective 5-HT Detection 4

serotonin include anxiety, fatigue, increased forgetfulness, low self-esteem, poor control of

impulses, and loss of pleasure in previously enjoyed activities.3 Beyond these symptoms, a

deficiency of serotonin can have drastic medical effects. According to the world health

organization, approximately 6.7 percent of the U.S. population of adults suffers from

depression.4 Approximately 10 percent of Americans over age 65 will be diagnosed with

Alzheimer’s.4 It is thus not a rarity for a deficit of serotonin to make an immense impact on our

lives. These affects occur when serotonin levels are below the range of 101-283 ng/ml. However,

beyond this range, other diseases may result, such as carcinoid syndrome, a type of cancer in

which tumors release excess serotonin to the bloodstream and cause symptoms such as rapid

heartbeat and difficulty breathing.5 Acknowledging the plethora of symptoms and disorders that

potentially result from a deficit or excess of serotonin, it is not surprising that a large area of

current research focuses on detecting levels of this key neurotransmitter.

2. Review of Literature

2.1. 5-HT linked to depression

In 1976, Asberg and a team of researchers measured the concentration of a metabolite of

serotonin in 68 patients suffering from depression.6 They discovered a negative correlation

between the concentration of this form of serotonin and the severity of the depression, meaning

that lower levels of serotonin were related to more severe depression. Today, the most common

medications to treat depression are known as selective serotonin reuptake inhibitors (SSRIs).6

These work by reducing the rate at which serotonin released into the brain is reabsorbed into

cells, increasing the overall level of serotonin in the extracellular space.6 This indicates that

serotonin does indeed play a role in diseases such as depression, but also that there is hope in the
Selective 5-HT Detection 5

form of treatment. This treatment must start at detection. There are numerous researched studies

that report approaches to neurotransmitter detection. While the plan throughout this year

remained to develop a mechanism of serotonin detection, my approach changed due to the

inherent fluctuating nature of research.

2.2. Cyclic voltammetry

After analyzing the case study by Asberg et al. to develop a deeper understanding of the

implications of a serotonin deficit, several mechanisms for serotonin monitoring were studied in

attempts to ascertain which is most effective in identifying low levels of serotonin. Research by

R. Mark Wightman and Elizabeth S. Buchner focusing on cyclic voltammetry was utilized as a

model to develop a technique to electrochemically detect serotonin.7 Voltammetry looks at

specific voltages where molecules become oxidized or reduced and thus exchange electrons in

redox reactions. This technique involves applying a triangular waveform to a microelectrode at a

high scan rate to oxidize and reduce the electroactive species at the surface of the electrode. Peak

currents are converted into concentrations using calibration factors from standards of known

concentration. Cyclic voltammetry usually works even to the extent to monitor neurotransmitters

in freely moving animals.7 Thus, this technique presented an opportunity to utilize

microelectrodes available at Roberts Wesleyan College to first detect serotonin in solution, move

on to lysed cells and test the technique in vitro, and finally perform detection assays in vivo.

Unfortunately, in order to detect serotonin at biological levels, fast-scanning ultramicroelectrodes

are needed, and our specific instrumentation did not reach these speeds or detection limits,

leading to the pursuit of alternative detection methods.

Selective 5-HT Detection 6

2.3. Aptamer-Gold Nanoparticles

Aptamer research was modeled after the study titled, “Fast and Selective Plasmonic

Serotonin Detection with Aptamer-Gold Nanoparticle Conjugates,” conducted by Chávez et al.8

This team researched serotonin detection because it is known that the neurotransmitter is linked

to a number of conditions, yet a full understanding of its role in these diseases is lacking

significantly. The development of fast, selective detection methods will provide them the tools to

monitor serotonin in patients both before and after treatment for specific conditions.8 It was their

goal to develop a fast assay for point-of-care and personalized diagnostics applications that

would not be compromised in the presence of biofluids in vivo.8 Many techniques utilized prior

to this study were based upon high-performance liquid chromatography (HPLC) separation of

serotonin present in whole blood coupled to detection methods such as tandem mass

spectrometry (MS-MS).8 While techniques of this nature have indeed provided platforms for

precise and accurate serotonin quantification, they had inherent drawbacks inhibiting the desired

rapid diagnostic ability, requiring specialized instrumentation, and being time-consuming.8 Thus,

a technique to rapidly monitor serotonin levels in whole blood using low volumes of sample and

requiring minimum sample treatment would be of immense clinical value. Through the

combination of gold nanoparticles with aptamers, Chávez et al. were able to design a number of

successful colorimetric sensors for different targets that have revolutionized the field of

diagnostics.8 These assays for serotonin were based on the use of aptamer-gold nanoparticle

conjugates utilizing small sample volumes, short response time, and an easily detectable

colorimetric output capable of being coupled to portable devices such as tablets and smartphones

for point-of-care testing.8

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2.4. AuNPs, N-acetyl-L-cysteine, DSP

The second colorimetric technique analyzed was modeled after work by Godoy-Reyes et

al. titled, “Selective and Sensitive Colorimetric Detection of the Neurotransmitter Serotonin

based on the Aggregation of Bifunctionalised Gold Nanoparticles.”9 This article reports a simple,

selective, and sensitive method for colorimetric detection of serotonin in aqueous media using

gold nanoparticles, dithiobis(succinimidylpropionate)(DSP) and N-acetyl-L-cysteine (NALC).9

A colorimetric change from red to blue capable of being observed with the naked eye was

induced due to a double interaction between nanoparticles and the hydroxyl and the amino group

of serotonin leading to an interparticle-crosslinking aggregation.9 Through the use of other

neurotransmitters including dopamine, epinephrine, and norepinephrine, certain biomolecules

including gamma-aminobutyric acid, glutamic acid, and aspartic acid, as well as common

inorganic species as controls, it was determined that this colorimetric detection method was

selective to 5-HT as other neurotransmitters tested as controls did not illicit a color change. 9

Utilizing UV-vis titrations, a limit of detection of 0.1 µM in buffered water was observed.9

Similarly, when the probe was utilized in simulated blood serum, a limit of detection of 0.12 µM

and a linear response within the 0-3 µM concentration range were observed, notable as these fall

within the range of the 5-HT concentrations of clinical interest.9 Finally, this technique was

tested in real human blood samples and proved to provide a remarkably accurate method to

distinguish between normal 5-HT levels and those indicative of disease.9 Given that this

technique required chemicals already included in Roberts Wesleyan resources without additional

expenditures on aptamers and was characterized by a colorimetric quantification method, it

became attractive as a technique to stem from and build upon. Thus this technique was selected

as one to model research after in order to detect serotonin to help elucidate the role played by this
Selective 5-HT Detection 8

neurotransmitter and develop innovative mechanisms for personalized diagnosis applications.

3. Methodology and Findings

3.1. AuNPs Preparation

After a thorough analysis of current research techniques utilized to detect levels of

neurotransmitters and preliminary experimental results at Roberts Wesleyan College, two

colorimetric techniques were analyzed to detect serotonin. Both of these techniques began with

the preparation of gold nanoparticles, or clusters of gold atoms in a spherical crystal structure.

These particles are utilized in an incredibly broad range of applications due to their unique

physical and chemical properties. Some examples include using them for target drug delivery,

for detection of cancer cells, and for genome therapy. When particles are not formed correctly,

they precipitate from solution and remain in clumps on the bottom of the flask. This aggregation

process leaves the color of the solution blue. Gold nanoparticles were made by combing 300 µL

of 10% sodium citrate and 81 µL of 17% gold chloride to 80 mL deionized water that was boiled

and stirred for 10 minutes until the solution changed color from a pale purple to a vivid and deep

ruby. Sodium citrate was utilized to keep the particles suspended in solution. When proper

reactant concentrations were utilized, the particles remained in the solution producing the ruby

color. Through analysis via UV-vis spectroscopy, it was determined that the synthesized gold

nanoparticles had a wavelength at maximum absorbance of 518 nm, which is indicative of a 10-

12 nm in diameter nanoparticle. Alternatively, AuNPs were produced from 0.2 M potassium

carbonate, 9.9% gold chloride and water at 4 °C having diameters between 2 and 5 nm, but the

10 nm size were found to be most advantageous and were utilized in most of the studies.

Because future experimentation would require attaching expensive molecules, such as

aptamers, to the gold nanoparticles, it was necessary to experiment with the interactions between
Selective 5-HT Detection 9

polyethylene glycol (PEG) and AuNPs to develop a consistent technique of attaching molecules

to these particles. The form utilized was a thiolated compound with a sulfur group capable of

forming strong interactions with the gold surface. PEG is thus known for its ability to keep

AuNPs from falling out of solution and for enhancing their potential advantages, as explored by

Manson et al.10

3.2. Apt-AuNPs-Serotonin Assay

The first colorimetric test utilized to quantitatively monitor the presence of serotonin

involved aptamer-conjugated nanoparticles. Aptamers are short DNA sequences with a name

derived from the latin, “aptus,” meaning “to fit,” and the greek, “meros,” meaning, “part.”11

Thus, the name aptamer literally refers to the ability to bind by fitting with a particular part of a

target.11 As part of the first technique to detect serotonin, aptamers were utilized for their binding

abilities to initially bind to one particle and later unfold and bind to another, producing a

conformation change that resulted in a color change. The aptamers were prepared for

experimentation by centrifuging the aptamer tube to ensure the dried pellet was at the bottom of

the container before resuspending it with 1 mL of resuspension buffer. At this point, 1000 µL of

aptamer solution were available for experimentation. For each trial, 50 µL of the aptamer

solution was resuspended in 100 µL of folding buffer and heated for 5 minutes at 95 °C to unfold

the DNA aptamer and allow it to refold properly. Precisely 1000 µL of gold nanoparticles were

added to this solution, and 750 µL aliquots of this final solution were added to each of 4 vials to

be utilized in colorimetric experimentation with varying concentrations of serotonin, as observed

in Table 1. Here, it is evident that for each trial, several vials were prepared with increasing

concentrations of serotonin in order to observe a color change with increasing intensity. It should
Selective 5-HT Detection 10

be noted that Vial 2 contained the concentration of serotonin in the range of clinically healthy

serotonin levels.

Table 1: Data table depicting experimental set-up of colorimetric assay with aptamers

Vial: 1 2 3 4

Volume of Apt/AuNP sol’n (µL) 750 750 750 750

Volume of 5-HT (µL) 0 2.5 5 10

Concentration of 5-HT (ng/mL) 0 200 2500 10,000

Volume of D.I. H2O (µL) 10 7.5 5 0

3.3. Apt-AuNPs-Serotonin Results

The exceptional advantages of aptamers come in to play as they unfold from their bound

position with the GNPs and refold with serotonin to produce a colorimetric result. Figure 2

shows that as the concentration of serotonin increased, a more distinct change in color was

observed. The wells on the bottom of this plate correspond to gold nanoparticles in solution with

aptamers presented with dopamine instead of serotonin as a control. According to research done

by Chavez et al., when similar aptamers were utilized to detect serotonin, color changes from

purple to pink were observed.8 However, according to this particular figure from my

experimentation, consistent with innumerable other trials, our data indicate that the presence of

serotonin induced a color change directly opposite, from pink to purple with increasing

concentration. Thus, the results of our research with aptamers remained inconclusive.
Selective 5-HT Detection 11

Figure 2: Depiction of color change underwent by GNP-aptamer solution in the presence of

serotonin (top) and dopamine (bottom).

3.4. AuNPs, N-acetyl-L-cysteine, DSP, serotonin assay

The second colorimetric technique examined to detect serotonin involved a color change

in solution to indicate the presence of the neurotransmitter. DTT is a powerful reducing agent

that reduces disulfide bonds by losing hydrogen atoms to result in its oxidized forms and adding

hydrogen atoms to other compounds to result in their reduced forms. As observed in Figure 3,

DSP has a disulfide bond, which is capable of being reduced by DTT. N-acetyl-L-cysteine

(NALC) is the N-acetyl derivative of the amino acid L-cysteine. As observed in its structure, it

has a sulfhydryl (SH) group that is able to reduce free radicals. This makes it an extremely

popular drug used as a mucolytic agent to relieve coughing and thick phlegm, as well as to

reduce anxiety.12

DSP →3,3’-dithiodipropionic acid di (N-hydroxysuccinimide ester)

Selective 5-HT Detection 12

DTT→ dithiothreitol

NALCàN-acetyl-L-cysteine

Figure 3: Structures of chemicals utilized in serotonin detection method

To carry out this colorimetric test, NALC and DSP were added to separate vials of gold

nanoparticles. In varying trials, differing concentrations of DSP and cysteine were added to the

gold nanoparticles to ascertain the optimum number of molecules of each necessary to account

for the gold particles present. Finally, aliquots of each separate solution were combined and

presented with serotonin to induce a change in color from pink to blue indicating the presence of

the neurotransmitter. An alternative method that provided more consistent results involved the

production of solutions containing both DSP and NALC bound to the same nanoparticles rather

than being bound to separate solutions and combined after. Specifically, 50 µL of 5 x10-4 M DSP

and NALC were added to each of 4 vials containing 1000 µL of AuNPs as observed in Table 2.

Increasing concentrations of 5-HT were added to observe a colorimetric change upon the

aggregation of serotonin. Also of note, the concentration range of serotonin present in clinically

healthy blood levels was included. Control experiments also included combining unmodified
Selective 5-HT Detection 13

AuNPs with serotonin without DSP and NALC and combing AuNPs, DSP, NALC, and D.I. H2O

without 5-HT.

Table 2: Data table depicting experimental set-up of colorimetric assay with DSP and NALC

Vial: 1 2 3 4

Volume of GNP sol’n (µL) 1000 1000 1000 1000

Volume of DSP (µL) 50 50 50 50

Volume of NALC (µL) 50 50 50 50

Volume of 5-HT (µL) 0 15 30 50

Concentration of 5-HT 0 200 2500 10,00

(ng/mL) 0

Volume of D.I. H2O (µL) 10 7.5 5 0

3.5. AuNPs, N-acetyl-L-cysteine, DSP, serotonin results

AuNPs, NALC, and DSP combine to create a colorimetric test to detect serotonin as seen in

Figure 4. When DTT is in solution with DSP, it reduces DSP’s disulfide bonds to produce the

reduced form having a sulfhydryl group. Gold nanoparticles form strong interactions with sulfur

groups, explaining the binding of the reduced form of DSP and N-acetyl-L-cysteine to the gold

nanoparticles. When DSP and Cysteine are both in solution with gold nanoparticles, the solution

is initially pink/red. When the original pink solution comes contact with serotonin, hydrogen

bonds form between serotonin and the cysteine molecules bound to the gold nanoparticles, and

covalent bonds form between DSP molecules and serotonin. This is possible because serotonin

contains a terminal amine and hydroxyl group, and the nitrogen, oxygen, and hydrogen atoms
Selective 5-HT Detection 14

will hydrogen bond with cysteine. This produces a blue color in the solution due to the

aggregation to indicate the presence of serotonin.

Figure 4: Chemical reaction in which the presence of serotonin induces the formation of
hydrogen bonds between N-acetyl-L-cysteine and serotonin to produce a color-changing
aggregation.9

Once DSP, cysteine, and gold nanoparticles were in solution together, the result was

centrifuged, or spun extremely rapidly, to reveal a clear/red liquid above a deep ruby soft pellet,

as pictured in Figure 5. This purification procedure was necessary to remove excess reagents.

When the top layer was removed, the pellet was resuspended into a buffer solution and serotonin

was added in varying amounts. As pictured, it is upon the addition of serotonin that a color

change from red to blue is observed, indicating the presence of serotonin. Figure 5 depicts the

process of change in color from when gold nanoparticles, DSP, and NALC are initially in

solution, to when they have been centrifuged so that only a pellet containing nanoparticles bound

with DSP and NALC remains, to the pink color of this pellet when brought back into solution

with a buffer to finally the purple tint when serotonin is introduced to the particles. The control
Selective 5-HT Detection 15

experiment combining solely AuNPs with serotonin without DSP and NALC ultimately

aggregated with the AuNPs which fell out of solution and that combing AuNPs, DSP, NALC,

and D.I. H2O without 5-HT remained pink in color.

GNP/DSP/NALC
Pellet and Pellet
Pellet aggregation
excess resuspension with 5-HT
GNP/DSP/NALC
DSP/NALC

0 200 2,500 10,000

ng/mL ng/mL ng/mL ng/mL

Figure 5: Photographic evidence of the purification process (top) and increasing concentrations
of serotonin with color change of increasing intensity

3.6. UV-vis Spectroscopy

UV-vis spectroscopy is an instrumental technique that involves a light source shining

onto a grating and being separated into its component wavelengths. Each single wavelength
Selective 5-HT Detection 16

beam passes through small containers of a sample, and the intensity of the light is measured both

before entering and after exiting the sample. The difference in intensity of light is indicative of

how much light was absorbed by the sample and what particular wavelengths were absorbed.13

When gold nanoparticles are in solution with DSP and cysteine, a peak is observed around 525

nm as shown in Figure 6, characteristic of the specific surface plasmon band formed when

surface electrons on particles are excited by UV light.9 Specifically, peaks arise in UV-vis

absorption spectras when electrons from the highest occupied molecular orbital (HOMO) level

absorb appropriate energy in the form of UV-vis light satisfying selection rules and are excited to

a higher unfilled shell. When serotonin is present, the process of aggregation occurs, shifting the

location of the plasmon band to 610 nm. This shift occurs from a shorter (525nm) to longer (610

nm) wavelength because when serotonin binds to the particles, a larger (longer) aggregate is

formed. Figure 6 depicts the UV-vis spectra produced after analyzing a series of five

concentrations of serotonin. As the concentration of serotonin in solution increases, a decrease in

intensity of the peak at 525 nm and an increase in intensity of the peak at 610 nm is observed.

Most notably, this method was tested utilizing a concentration of serotonin within the range of

normal levels in the blood, at 200 ng/mL, graphically depicted by a purple line in Figure 6.

Because this low concentration of serotonin was still detectable using this method, we are

confident that this technique could accurately be applied to cell samples in vivo. Through

experimentation with the goal of obtaining the minimum detection limit, serotonin was detected

at concentrations of above 120 ng/mL. It should be noted that an ideal UV-vis spectra produced

from analysis of these samples should depict a complete shift with the disappearance of the peak

at 525 nm and the appearance of the peak at 610 nm, indicating that further optimization

experiments are necessary.

Selective 5-HT Detection 17

Figure 6: UV-vis Spectra indicating the shift in the surface plasmon band indicating the presence
of serotonin

4. Conclusions

The development of a viable technique to monitor serotonin and the verification of its

efficacy in various laboratory settings including in stock solutions, in vitro, and in vivo, is

undoubtedly a process requiring extensive time and resources. Over the course of approximately

one year of research, it was possible to analyze an array of current research to perform

preliminary experiments to determine a general area of chemistry instrumentation to specialize in

with this specific research. After discerning that colorimetric tests were viable with Roberts

Wesleyan College resources, two discrete techniques were analyzed in parallel to discern which

provided most consistent results. The chosen technique involved the production of gold
Selective 5-HT Detection 18

nanoparticles and the interactions between these particles with DSP and NALC to produce

molecules capable of forming hydrogen bonds with serotonin and aggregating to indicate its

presence. After optimizing this technique, consecutively lower concentrations of serotonin were

detected to ascertain the detection limit of the method. Ultimately, this method was effective in

detecting 5-HT at concentrations above 120 ng/mL.

Once an optimized technique has been developed to monitor serotonin within this

concentration range, a mast cell line may be produced in which cells will be lysed, or flushed

with solution so that osmosis occurs to destroy the cell membrane to reveal the contents.

Specifically, the resources at Roberts allow for HaCat cells to be utilized and lysed. HaCat cells

are utilized because they are an immortal cell line that can replicate infinitely and are known for

being robust.14 When 5-HT is added to the contents, the selected detection technique will be

utilized to confirm its ability to detect in cells as opposed to in solution. Beyond in vitro

research, future work includes the creation of a laboratory for mice to be able to monitor

serotonin in vivo by extracting cells in the least invasive ways to assert the NALC method is

viable in vivo. From this point, it would be possible to experiment with a variety of physiological

tests applied to mice to see exactly what increases and decreases the levels of serotonin.

In addition to moving forward with the experimentation to detect serotonin in vivo, future

work also includes optimization studies to produce more favorable results. For example, the

detection method utilizing aptamers could be significantly improved with the use of thiolated

aptamers, while unfortunately greatly increasing expenditures.15 Furthermore, in their paper

titled, “Citrate-capped Silver Nanoparticles as a Probe for Sensitive and Selective Colorimetric

and Spectrophotometric Sensing of Creatinine in Human Urine,” Alula et al. demonstrate that

silver nanoparticles show promise in obtaining lower detection limits than gold nanoparticles.16
Selective 5-HT Detection 19

This means that if utilized in the cysteine method, serotonin could be detected when present in

even smaller concentrations. Ideally, the lowest detectable concentration of serotonin should be

below that found in normal blood. Thus, with these advancements, it should be possible to

accurately and reliably detect the lowest concentrations of serotonin found in blood. It has been

established that serotonin is a critical neurotransmitter to human metabolic regulation, so being

able to effectively monitor its current level in the body and knowing how to regulate it is of the

utmost importance.

.
Selective 5-HT Detection 20

References

1. Mukherjee, S., Seal, M. & Dey, S.G. Kinetics of serotonin oxidation by heme–Aβ relevant to
Alzheimer’s disease. Journal of Biological Inorganic Chemistry. 2014, 19, 1355-1365.

2. 2017 Alzheimer’s Disease Facts and Figures. Alz.org. https://www.alz.org/facts/ (accessed

November 2, 2017).

3. Brain Basics https://www.nimh.nih.gov/health/educational-resources/brain-basics/brain-

basics.shtml (accessed Jul 14, 2017).

4. World Health Organization 2018. Available at:

http://www.who.int/mediacentre/factsheets/fs362/en/. Accessed April 6, 2018.

5. Psychologistworld.com 2018. Available at:

https://www.psychologistworld.com/biological/neurotransmitters/serotonin. Accessed April 6,
2018.

6. Asberg M, Thoren P, Traskman L et al. "Serotonin depression"--a biochemical subgroup

within the affective disorders? Science; 1976;191:478-480.

7. Bucher, E.; Wightman, R. Electrochemical analysis of neurotransmitters. Annual Review of

Analytical Chemistry 2015, 8, 239-261.

8. Chávez J, Hagen J, Kelley-Loughnane N. Fast and Selective Plasmonic Serotonin Detection

with Aptamer-Gold Nanoparticle Conjugates. Sensors; 2017;17:681.

9. Godoy-Reyes T, Llopis-Lorente A, Costero A et al. Selective and sensitive colorimetric

detection of the neurotransmitter serotonin based on the aggregation of bifunctionalised gold
nanoparticles. Sensors and Actuators B: Chemical; 2018;258:829-835.
Selective 5-HT Detection 21

10. Manson J, Kumar D, Meenan B, Dixon D. Erratum to: Polyethylene glycol functionalized
gold nanoparticles: the influence of capping density on stability in various media. Gold Bulletin.;
2011;44:195-196.

11. Base Pair Biotechnologies 2018. Available at: https://www.basepairbio.com/what-is-an-

aptamer/. Accessed April 4, 2018.

12. Frank K, Patel K, Lopez G, Willis B. Examine.com 2018. Available at:

https://examine.com/supplements/n-acetylcysteine/. Accessed April 4, 2018.

13. BiosensingUSA.com 2018. Available at: http://biosensingusa.com/technologies/surface-

plasmon-resonance/surface-plasmon-resonance-work/. Accessed April 4, 2018.

14.Seo M, Kang T, Lee C et al. HaCaT Keratinocytes and Primary Epidermal Keratinocytes
Have Different Transcriptional Profiles of Cornified Envelope-Associated Genes to T Helper
Cell Cytokines. Biomolecules and Therapeutics; 2012;20:171-176.

15. Jeong S, Kim C, Yang J. Comparison of the sensitivity of thiolated aptamer based biosensor
according to the condition of electrode substrates. BioChip Journal; 2010;4:141-147.

16. Alula M, Karamchand L, Hendricks N, Blackburn J. Citrate-capped silver nanoparticles as a

probe for sensitive and selective colorimetric and spectrophotometric sensing of creatinine in
human urine. Analytica Chimica Acta; 2018; 1007:40-49.