CHAPTER ONE

1.0 Introduction
1.1 Background and Significance
One of the most common cancers impacting men worldwide is prostate cancer. For successful
treatment and better patient outcomes, prostate cancer must be accurately detected early and
monitored throughout time. Due to its link to prostate pathology, prostate-specific antigen (PSA)
has been widely employed as a biomarker for prostate cancer screening and monitoring.
The prostate gland produces the glycoprotein enzyme PSA, which is essential for the liquefaction
of semen. Elevated PSA levels can be a sign of several prostate disorders, including prostate
cancer, benign prostatic hyperplasia (BPH), and prostatitis. It is discharged into the bloodstream.
It is crucial to remember that elevated PSA values do not always indicate prostate cancer and that
additional diagnostic procedures, including a prostate biopsy, are required for a certain diagnosis.
Enzyme-linked immunosorbent assays (ELISAs), which are serum-based, are the conventional
method for determining PSA levels. Although ELISA is frequently used, it takes a lot of time,
involves complicated sample preparation, and may not have the sensitivity and selectivity needed
for reliable prostate cancer detection. This has prompted extensive research to create alternative
techniques, including biosensors, for PSA detection.
A walnut-sized organ, the prostate gland is in front of the rectum, just below the bladder. The
urethra, the passageway via which urine and sperm exit the body, is encircled by it. A tiny
amount of PSA normally leaks into the bloodstream, but elevated PSA levels might signify a few
diseases, including prostate cancer, benign prostatic hyperplasia (BPH), and prostatitis (prostate
inflammation).
One of the most common types of cancer among males is prostate cancer. Because elevated PSA
levels may indicate the existence of cancer cells in the prostate gland, the PSA test is useful in
identifying early signs of prostate cancer. The existence of cancer is not always indicated by a
high PSA test, it is crucial to remember this. PSA levels can also be impacted by other elements
like age, prostate size, and other medical problems.
A digital rectal exam (DRE) and a prostate biopsy may be advised if a PSA test reveals elevated
PSA levels to identify the reason of the elevated PSA and confirm or rule out prostate cancer.
These extra examinations give a more thorough picture of the prostate gland and aid in precise
diagnosis.

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It is crucial to realize that the PSA test has some limitations even if it is a useful screening tool.
PSA levels can be influenced by several non-cancer-related causes, and not all prostate cancers
will result in noticeably high PSA levels. Additionally, not every case of prostate cancer is
severe enough to need rapid medical attention. Therefore, a thorough assessment by a healthcare
expert that considers everyone's circumstances and preferences should be used to decide whether
to move forward with more tests or treatment.
For males over 50, routine prostate cancer screening, including PSA testing, is usually advised.
The precise recommendations, however, may change based on personal risk factors and medical
history. It is advisable to speak with a healthcare provider to decide on the best screening plan
and to go over the advantages and restrictions of PSA testing in particular circumstances.
The two main types of PSA blood tests are:

1. Total PSA Test: This test determines the total concentration of PSA in the blood,
including complex (bound to other proteins) and free PSA. A common initial prostate
cancer screening method is the total PSA test. Prostate cancer may be present if total PSA
levels are elevated, but more testing is typically required to make the diagnosis.
2. Free PSA Test: This test measures only the PSA that is unattached to other proteins. It
determines the proportion of free PSA there are overall. To determine the likelihood of
prostate cancer, this test is occasionally used with the total PSA test. A lower proportion
may indicate a higher risk, whereas a higher percentage generally indicates a lower risk
of prostate cancer.

1.2 Objective
This seminar's main goal is to examine PSA detection with manufactured electrodes, a potential
platform for sensitive and targeted PSA detection. Fabricated electrodes have several benefits
over conventional techniques, including simplicity in fabrication, possibility for downsizing,
quick response, and increased sensitivity.

1.3 Overview of Prostate Specific Antigen

The well-known cancer biomarker prostate-specific antigen (PSA) is frequently employed for
monitoring and diagnosing prostate cancer in its early stages (Parnaz Assari, et al., 2020). Given
that increased PSA levels are linked to the presence of the illness (Tian, et al., 2012), it is

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essential for the early detection and treatment of prostate cancer. The average PSA level in
healthy men is about 4 ng/mL (Tian, et al., 2012). For the clinical diagnosis and treatment of
prostate cancer, accurate determination of PSA levels in human serum is essential (Dey et al.,
2012).
Various techniques and strategies have been employed for the analysis of PSA, including surface
plasmon resonance (Uludag, et al., 2012), immunosensors, fluorescence (Huhtinen et al., 2004),
electrochemical aptasensors (Parnaz Assari, et al., 2020), mass spectrometry (Chen et al., 2014),
and electrochemical immunoassay (Parnaz Assari, et al., 2020). Among these techniques,
electrochemical immunoassay has gained dominance due to its high sensitivity, fast detection,
low cost, simplicity of instrumentation, and real-time monitoring capabilities (Goyal et al.,
2007).
A technique that shows promise for raising the analytical performance of PSA detection is the
label-free electrochemical immunosensor (Parnaz Assari, et al., 2020). Due to their inherent
benefits, such as a large surface area, high conductivity, and distinctive catalytic activities,
nanostructured materials, such as carbon nanotubes (CNTs) as stated by B. Prieto-Simón, (2015),
conducting polymers (CPs) according to Barton, 2008, and nanoparticles (Parnaz Assari, et al.,
2020), have attracted attention in the construction of immunosensors.
Due to their outstanding physicochemical characteristics, such as their extraordinary electrical
conductivity and electrochemical stability, multi-walled carbon nanotubes (MWCNTs) have
attracted particular attention (Parnaz Assari, et al., 2020). Conducting polymers, including
polyaniline (PANI), have been used frequently as electrode modification sensing substrates
(Karimi-Maleh et al., 2015). PANI stands out in particular because of its strong biocompatibility,
environmental stability, and simplicity of manufacture (Parnaz Assari, et al., 2020).
Additionally, it has been demonstrated that the addition of carboxylic acid groups to MWCNTs
(COOH-MWCNTs) can facilitate the polymerization of aniline, resulting in a composite matrix
that combines the mechanical strength and electric conductivity of the polymer (Parnaz Assari,
et al., 2020).
To achieve the necessary sensitivity for PSA detection, especially in real samples that are
typically diluted, highly sensitive procedures (less than ng·mL−1) are required (Akter et al.,
2012). The incorporation of gold nanoparticles (AuNPs) in immunosensors has proven
beneficial, as they promote electron transfer reactions, possess excellent conduction

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characteristics, enable simple operation, and exhibit biocompatibility with proteins (Afraz et al.,
2014). AuNPs also serve as ideal hosts for the immobilization of antibodies due to their
combination of physical and chemical properties (Parnaz Assari, et al., 2020).
For better prostate cancer detection, it is essential to develop new biomarkers and non-invasive
diagnostic techniques (Landers, et al., 2005). Prostate cancer gene 3 (PCA3), a long noncoding
RNA generated only in prostate cancer tissues, is one such biomarker (Parnaz Assari, et al.,
2020). Prostate cancer diagnoses have shown better specificity when PCA3 and PSA indicators
are combined (Parnaz Assari, et al., 2020). The Progensa PCA3 test has been shown to be
successful in minimizing unnecessary prostate biopsies (Parnaz Assari, et al., 2020). It is based
on urine samples and uses RT-PCR to determine the PCA3 lncRNA/PSA mRNA ratio. However,
many communities continue to find this test to be expensive and unavailable (Parnaz Assari, et
al., 2020).
A potential method for the detection of PCA3 uses nucleic acid-based bioreceptors in
conjunction with electrochemical biosensors (Parnaz Assari, et al., 2020). Because of their
excellent selectivity, sensitivity, quick reaction, and mobility, these biosensors, which use
screen-printed electrode biochips, are appropriate for point-of-care diagnostics (Parnaz Assari, et
al., 2020). Using redox-labeled aptamers, recent studies have concentrated on creating
electrochemical biosensors for PCA3 detection (Parnaz Assari, et al., 2020). These biosensors
have been proven to have low limits of detection and are promising for finding PCA3 residues in
common urine samples (Parnaz Assari, et al., 2020). High sensitivity has been attained using
methods like cyclic voltammograms (CV), differential pulse voltammograms (DPV), and
electrochemical impedance spectroscopy (EIS) (Parnaz Assari, et al., 2020). The detection of
PCA3 may be improved with further study of DPV, which is well known for its outstanding
sensitivity in monitoring medical components (Parnaz Assari, et al., 2020).
The utilization of electrochemical biosensors combined with nucleic-acid-based bioreceptors has
shown promise for the detection of PCA3, an important biomarker in prostate cancer diagnostics.
These biosensors offer high selectivity, sensitivity, and portability, making them suitable for
point-of-care applications. Future advancements in electrochemical biosensing techniques, such
as DPV, hold potential for further improving the detection of PCA3 and enhancing the early
diagnosis of prostate cancer.

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In conclusion, electrochemical immunosensors have attracted a lot of attention for their potential
in the early detection and monitoring of prostate cancer. The use of nanostructured substances,
such as COOH-MWCNTs, PANI, and AuNPs, has the potential to enhance the sensitivity and
effectiveness of the immunosensor. These developments help support ongoing efforts to better
understand and treat prostate cancer.

1.4 Importance of PSA Detection

Prostate cancer early detection and screening:
Prostate cancer early detection and screening: PSA is a sensitive marker for prostate cancer early
detection. PSA blood levels that are elevated can be a sign of prostate cancer cells. It's crucial to
remember that PSA cannot accurately identify prostate cancer on its own because raised PSA
levels can also result from other conditions including prostatitis and benign prostatic hyperplasia
(BPH). Traditional PSA cutoff values are 4 ng/mL, and other diagnostic procedures, such as
ultrasound-guided biopsy, are frequently carried out to confirm the presence of cancer
(Oesterling, et al., 1991).
Prostate Cancer Monitoring:
PSA levels in the blood correlate with the volume of prostate cancer and provide valuable
information about the disease process. Different types of prostatic tissue contribute differently to
serum PSA levels. On average, the normal prostate contributes 0.2 ng/mL per gram of tissue,
BPH tissue contributes 0.6 ng/mL, and cancerous tissue contributes approximately 2 ng/mL.
Therefore, even relatively small tumors (0.3-1 mL) can cause an elevation in serum PSA.
However, there is considerable variation between patients (Stamey et al., 1987).
Serum PSA levels decrease following prostate cancer treatment that is effective. The sort of
therapy used determines the rate of decline. After a radical prostatectomy, PSA levels quickly go
undetectable, indicating that malignant tissue has been removed. The existence of residual or
recurring cancer cells is indicated by a prolonged rise in PSA levels beyond the detection limit,
which is an early symptom of relapse. Relapses can be identified using ultrasensitive PSA testing
months or even years earlier than with traditional assays, enabling prompt intervention and
therapy (Ulf-Hakman, et al., 1999).

PSA and Treatment Response:

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PSA is a trustworthy predictor of how prostate cancer patients will respond to hormone therapy.
Because androgens control how PSA expresses, hormonal therapy that suppresses the PSA gene
causes a drop in serum PSA levels. Monitoring PSA levels during hormone therapy aids in
determining the efficacy of the medication and the direction of the disease (Ulf-Hakman, et al.,
1999).
PSA is important in clinical practice because it helps with early detection, monitoring, and
evaluation of therapy response in patients with prostate cancer. It makes it possible to recognize
those who are at risk, enabling prompt intervention and potentially curative therapy. By enabling
early detection of clinically organ-confined prostate tumors, which have a higher likelihood of
responding to therapy, PSA testing, together with other diagnostic techniques, helps to improve
patient outcomes (Ulf-Hakman, et al., 1999).

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CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 Overview of Biosensors for PSA Detection
Due to their capacity to combine biological recognition components with transducers, which
enables the conversion of specific biomolecular interactions into quantifiable electronic signals,
biosensors present a promising method for the detection of PSA. PSA biosensors can deliver
quick, accurate, and in-the-moment analysis, providing crucial information for the detection and
management of prostate cancer.
Biosensors can be classified based on the type of receptors used in bio-recognition events and the
type of transducers involved.

1. Receptor-Based Classification: Different types of receptors have been employed in

biosensors for PSA detection, including:

Antibody-Based Biosensors: Antibodies specific to PSA are immobilized on the sensor

surface, facilitating the recognition and binding of PSA molecules, leading to a
measurable signal (Altintas, et al., 2018). Antibody-based biosensors are widely used in
immunoassays for the detection and quantification of various analytes. These biosensors
typically involve the immobilization of antibodies or their derivatives onto the sensitive
surface of the sensor. The choice of immobilization method depends on the nature of the
antigen and the desired performance of the biosensor.
When immobilizing antibodies, the goal is to achieve maximum response in relation to
the analyte concentration in the sample, thereby achieving low detection limits. It is
crucial to immobilize the antibodies compactly on the sensor surface without altering the
structure of the antibody's active site or limiting its availability to interact with the
antigen. It is undesirable to employ methods that enhance antigen-binding capacity but
reduce the detected signal, such as forming additional layers that hinder the conductivity
of electrochemical sensors. In some cases, detecting immune complexes without

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separating bound and unbound labels can be achieved by spatially converging the label
and the sensitive surface.
The simplest method for immobilizing antibodies is physical adsorption, where the
antibodies are absorbed onto the sensor surface. However, this process can lead to
denaturation or significant conformational changes in the antibodies. An alternative
approach, commonly employed in numerous developments, is covalent coupling of
antibodies to a chemically activated sensor surface. Random coupling methods direct the
antibodies against the surface based on the reactive group of the antibodies in contact
with it.
To address the challenges associated with antibody immobilization, oriented
immobilization techniques have been successfully utilized. Oriented immobilization
ensures a unified antibody orientation, resulting in more reproducible characteristics of
the biosensor. Various approaches can be used to immobilize antibodies with chosen
orientation.
One approach involves utilizing cysteines present in the immunoglobulin molecule,
which are considerably distant from the antigen-binding site. This method includes direct
chemical conjugation of IgG with a sensor surface coated with dextran or a thiol layer.
Fab fragments of antibodies obtained by reducing Fab2 fragments can be immobilized on
thiol surfaces even more easily than native antibodies. Recombinant antibodies
containing C-terminal cysteine residues have also been effectively used for oriented
immobilization.
Indirect binding methods for oriented immobilization involve pre-forming an antibody-
binding layer on the sensor surface. Proteins such as staphylococcal protein A,
streptococcal protein G, or (strept)avidin coupled to biotinylated antibodies can facilitate
this orientation. In some cases, interactions between antibodies and antigens or protein A
occur in a homogeneous solution, followed by the transfer of oriented immune complexes
onto the sensor surface using electrostatic interactions between oppositely charged
polymers. The use of oppositely charged polyelectrolytes facilitates rapid separation of
immunoreactants from the reaction mixture.
The use of oriented immobilization techniques significantly improves the limit of
detection achieved by antibody-based biosensors. However, the extent of improvement

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varies depending on the specific immunosensor and the antigen being detected. Further
research is needed to understand the mechanisms underlying these differences in
performance for various types of immunosensors.
Antibody-based biosensors are widely used in various fields, including medical
diagnostics, environmental monitoring, and food safety. These biosensors leverage the
high specificity and affinity of antibodies to detect and quantify target analytes with
remarkable sensitivity. Immobilization of antibodies on the biosensor surface is a critical
step that directly influences the performance and detection limits of these biosensors.
The immobilization of antibodies in biosensors is crucial for maintaining their structural
integrity and maximizing their binding capacity. Different immobilization methods have
been employed, including physical adsorption and covalent coupling. Physical adsorption
is a simple method where antibodies are absorbed onto the sensor surface. However, this
approach can lead to denaturation or conformational changes, potentially affecting the
antibody's binding affinity and stability (Saerens, et al., 2008).
To address these challenges, covalent coupling has emerged as a preferred method for
antibody immobilization. Covalent coupling ensures a stable attachment of antibodies to
the sensor surface, preventing their dissociation during subsequent assays. This approach
often involves chemically activating the sensor surface and then coupling the antibodies
through reactive groups present on their structure (Wink, et al., 1997).
In addition to the immobilization method, the orientation of the immobilized antibodies
plays a crucial role in the performance of antibody-based biosensors. Oriented
immobilization techniques aim to align the antibodies in a specific orientation to enhance
their antigen-binding properties and improve the reproducibility of the biosensor's
characteristics. Several strategies have been employed for oriented immobilization,
including utilizing limited cysteine residues present in the immunoglobulin molecule,
utilizing proteins such as staphylococcal protein A or streptococcal protein G, or
employing electrostatic interactions between oppositely charged polymers (Dzantiev et
al., 2013).
The binding affinity of antibodies also impacts the limit of detection in antibody-based
biosensors. Antibodies exhibit a range of dissociation constants (KD) depending on their
affinity for the target antigen. While high-affinity antibodies are desirable, there is a limit

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to their effectiveness. The "affinity ceiling" hypothesis suggests that the selection of
antibodies with extremely high affinity may be less effective due to factors such as the
half-life of antigen complex formation and the proliferation of B cells as (Dzantiev, et al.,
2013). Nonetheless, certain antibodies can exhibit exceptionally high binding constants
and form non-dissociating antigen-antibody complexes (Chmura, et al., 2001).
The sensitivity and detection limits of antibody-based biosensors are also influenced by
the assay format employed. Noncompetitive and competitive immunoassay formats are
commonly used, each with its own advantages and considerations. Noncompetitive
assays directly detect the formation of immune complexes, while competitive assays
measure the competition between the analyte in the sample and a labeled antigen for
binding to the immobilized antibodies (Jackson, et al., 1986). Signal amplification
methods, such as the use of metal nanoparticles or multilayer systems with antibodies and
aptamers, can further enhance the sensitivity of these biosensors (Mayer, et al., 2010).
Advancements have been made to detect extremely low concentrations of immune
complexes or even single interactions; such highly sensitive immunoassays often require
complex instrumentation and multi-step measurements (De La Rica, et al., 2021). The
use of antibodies with maximum affinity remains crucial for the development of highly
sensitive and widely applicable antibody-based biosensors (Jackson, et al., 1986).
Overall, antibody-based biosensors hold great promise for their ability to detect and
quantify target analytes with high sensitivity and specificity. Further research and
development in antibody immobilization, orientation techniques, and assay formats will
continue to advance the field, enabling the application of these biosensors in various
areas of science and technology.
Enzyme-Based Biosensors: Enzymes, such as horseradish peroxidase (HRP), are
utilized to catalyze reactions involving PSA, resulting in a signal amplification.
(Rocchitta, et al., 2016).
Most enzyme-based biosensors use certain enzymes that can identify and interact with
PSA molecules. Prostate-specific antigenase (PSAase), also known as kallikrein-related
peptidase 3 (KLK3), is a frequently utilized enzyme in PSA detection. In the presence of
PSA, PSAase can cleave peptide bonds.

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Enzyme-based biosensors for PSA detection work on the general idea of immobilizing
the enzyme on a surface, like a sensor electrode or a microfluidic channel. PSA
molecules bind to the immobilized enzyme when a sample containing PSA is added to
the biosensor. In the presence of PSA, the enzyme catalyzes a process that generates a
detectable signal.
Enzyme immobilization is a crucial technique in biotechnology that involves attaching
enzymes to a solid support or matrix, thereby enhancing their stability, reusability, and
ease of handling. Several methods have been developed for enzyme immobilization, each
offering distinct advantages and applications.
Enzymes are physically linked to the surface of a support material through weak contacts
such van der Waals forces, hydrophobic interactions, or electrostatic interactions in the
straightforward and frequently utilized process of adsorption. Silica, different resins, and
activated carbon are typical adsorption support materials. Adsorption is a relatively
simple procedure that doesn't involve significantly changing the enzyme or support
material chemically. However, leaching could limit the stability and reusability of the
enzyme immobilized through adsorption (Roberts, et al.,1992).
Covalent Binding: Covalent binding is the process by which an enzyme and a support
substance form durable covalent connections. With the help of this technique, enzyme
stability and retention on the support surface are improved. Covalent bonding can be
achieved via a variety of ways, such as surface activation methods like carbodiimide
chemistry, bifunctional reagents, or cross-linking agents. The type of enzyme and the
support material determine the approach to use. Covalent binding offers strong
immobilization, but because it involves chemical changes, it can occasionally change the
structure or activity of the enzyme (Zhiwei Zhao, et al., 2010).
Entrapment: Entrapment is the process of enclosing an enzyme in a porous support
substance, such as hydrogels, microspheres, or sol-gel matrices. As molecules of
substrate and products diffuse in and out of the support material's pores and holes, the
enzyme is physically imprisoned within these spaces. Entrapment offers enzyme
protection, preserves its native structure, and permits maximal enzyme loading. However,
there may be restrictions on the diffusion of substrates and products, which would result
in slower reaction rates (Mateo, et al., 2007).

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Cross-Linking: Cross-linking is the process by which covalent bonds are created between
the molecules of the enzymes, forming a three-dimensional network of immobilized
enzymes. To connect the enzymes, cross-linking chemicals like glutaraldehyde are
frequently utilized. Because cross-linking stops enzyme leaching, it improves stability
and reusability. Due to the production of stiff links, it can also result in enzyme
denaturation or reduced enzyme activity (Guisan, et al., 2006).
Encapsulation: Encapsulation is the process of enclosing an enzyme in a semipermeable
membrane or microcapsule that permits substrate and product diffusion while acting as a
protective barrier. Natural or artificial polymers can be used as encasing materials.
Excellent enzyme stability, defense against harsh environmental factors, and regulated
product release are all features of encapsulation. Encapsulation, however, may lead to
diffusion limits that impact the reaction rates (Kumar, et al., 2021).
Depending on the biosensor's design, the detectable signal may be electrical, optical, or
electrochemical. Once the signal has been measured and associated with the amount of
PSA present in the sample, PSA levels can be calculated.
High specificity, sensitivity, and comparatively low cost as compared to other detection
techniques are just a few benefits that enzyme-based biosensors offer in the detection of
PSA. These biosensors may be employed in clinical settings for the detection and
monitoring of prostate cancer in its early stages.
Aptamer-Based Biosensors: Synthetic oligonucleotide sequences called aptamers are
used to selectively bind to PSA, allowing for its detection (Grabowska, et al., 2017).
To detect a variety of target analytes with high sensitivity, specificity, and repeatability,
aptamer-based biosensors, often referred to as aptasensors, use aptamers as bioreceptors
(Cannatà et al., 2019). Aptamers are nucleic acid molecules having a molecular mass of
between 10 and 30 kDa that have 40 to 50 bases and can take the shape of either DNA or
single-stranded RNA (Yüce, et al., 2018).
Systematic Evolution of Ligands by Exponential Enrichment (SELEX), a method of
selecting compounds from random combinatorial libraries, is used to synthesis them in
vitro (Song, et al., 2008) Aptamers have several important benefits over antibodies,
including cost effectiveness, ease of modification and synthesis in vitro, high affinity,
great specificity, stability, and flexibility (Li, et al., 2008). Aptamers can be easily

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customized and functionalized with chemical changes, such thiol, biotin, or amino
groups, to make it easier for them to become immobilized on different surfaces and
operate as bioreceptors in biosensors (Evtugyn, et al., 2020).
Due to aptamers' high selectivity and affinity for their targets, the interaction between an
aptamer and its target analyte is like the interaction between antibodies and antigens
(Wang, et al., 2008). A variety of targets can be interacted with by aptamers thanks to
their distinctive and intricate three-dimensional structures, which include stems, loops,
bulges, hairpins, tetraloops, pseudoknots, kissing complexes, triplexes, and G-
quadruplexes (Mascini, et al., 2012). Hydrogen bonds, van der Waals forces, electrostatic
interactions, and stacking interactions can all be the basis for interactions between
aptamers and their target molecules (Mascini, et al., 2012).
An essential aspect of an aptamer is the dissociation constant (Kd), which expresses how
well the aptamer binds to the analyte. Lower Kd values denote more selective interactions
in aptamers, which have dissociation constants in the micromolar, nanomolar, and
picomolar ranges (Song, et al., 2008).Due to their high sensitivity, specificity, and
adaptability in detecting small molecules, heavy metals, pesticides, antibiotics, toxins,
and other analytes of interest, aptasensors have attracted significant attention in a variety
of fields, including medical diagnostics, environmental monitoring, and food safety
(Pietrantonio, et al., 2019).
In summary, aptamer-based biosensors, or aptasensors, leverage the unique properties of
aptamers as high-affinity bioreceptors for the detection of target analytes. By chemical
modifications and specific three-dimensional structures, aptamers offer several
advantages over traditional bioreceptors and have found extensive applications in
biosensing.

2.2 Application of Electrochemical Aptasensor for Virus Detection

Since Clark and Lyons created the first biosensor in 1962, which used enzyme
immobilization to detect blood glucose, the field of biosensor technology has made
considerable strides (Habibah, et al., 2016). Due to its multiple advantages over optical
and piezoelectric detection techniques, electrochemical aptasensors have attracted a lot of
attention recently for the purpose of detecting viruses. Electrochemical detection is a

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commonly used method for the detection of many biomolecules, including viruses
(Chand, et al., 2017), due to its portability, convenience of use, quick reaction, good
sensitivity, and high specificity (Labib et al., 2012). The aptasensor created by Chand and
Neethirajan to specifically detect Human Norovirus (HuNoV) is one illustration of an
electrochemical aptasensor created for viral detection (Chand, et al., 2017 ). The
aptasensor uses a particular viral aptamer and a modified graphene-gold nanoparticle
(Grp-AuNP) composite on a carbon electrode. The aptamer is biotin-modified, and
ferrocene molecules serve as redox probes to identify it. The Grp-AuNP composite
enables electrochemical signal amplification and offers a stable substrate for aptamer
immobilization. By binding to the norovirus capsid protein, the norovirus-specific
aptamer reduces the electrochemical signals from ferrocene. The aptasensor can also be
used to detect norovirus in blood samples, with a detection limit of 100 m (about 328.08
ft) obtained using the differential pulse voltammetry (DPV) electrochemical detection
approach (Chand, et al., 2017).
Another electrochemical aptasensor developed for virus detection targets avian influenza
virus type A H5N1 protein and was developed by Diba et al., 2015. This aptasensor
utilizes a sandwich system that involves the formation of an aptamer-protein-antibody
complex on gold nanoparticle (AuNP)-modified electrodes. The specific aptamer is
covalently immobilized on the surface of AuNPs, followed by the adsorption of the
H5N1 protein. The alkaline phosphatase (ALP) conjugated monoclonal antibody is then
adsorbed to form the AuNP-aptamer/H5N1/antiH5N1-ALP sandwich complex, which
binds to the surface of the electrode. The electrocatalytic reaction of ALP bound to 4-
amino phenyl phosphate (APP) substrate leads to an increase in the electrochemical
current, which is proportional to the H5N1 concentration. The DPV electrochemical
detection technique allowed the detection of H5N1 protein at a concentration as low as
100 fM (Diba, et al., 2015).
Additionally, Ghanbari et al., developed an electrochemical aptasensor for the detection
of the hepatitis C virus (HCV) antigen. HCV is a single positive RNA virus that primarily
infects hepatocytes in the liver. The aptasensor utilizes graphene quantum dots (GQD) as
a substrate for aptamer immobilization through π-π* stacking interactions, facilitated by
the amine group of the aptamer. The carboxyl groups of GQD enable strong adsorption of

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 the aptamer onto the GQD surface. The modification process and subsequent detection
steps employ electrochemical techniques such as electrochemical impedance
spectroscopy (EIS), cyclic voltammetry (CV), and DPV. The formation of the
aptamer/antigen complex leads to a significant increase in the electron transfer of the
redox ferricyanide K3[Fe(CN)6] system, which is detected using EIS.
The electrochemical aptasensor achieved a detection limit of 3.3 pg/mL-1 for HCV
antigen and accurately detected the antigen concentration in human serum samples
(Roushani, et al., 2017).
These examples demonstrate the application of electrochemical aptasensors for virus
detection. By leveraging the unique properties of aptamers and the advantages of
electrochemical detection, such aptasensors offer promising tools for rapid and sensitive
virus detection in various samples.

 Molecularly Imprinted Polymer (MIP)-Based Biosensors: MIPs are synthetic

polymers with specific binding sites designed to recognize and capture PSA
molecules with high affinity (Waffo et al., 2018).
2. Transducer-Based Classification: Various types of transducers are employed in PSA
biosensors, including:
 Electrochemical Biosensors: Electrochemical signals generated by the interaction
between PSA and the recognition element are measured using techniques such as
amperometry, voltammetry, or impedance spectroscopy (Mollarasouli, et al., 2019).
 Optical Biosensors: Optical techniques, such as surface plasmon resonance (SPR),
fluorescence, or bioluminescence, are utilized to detect changes in the optical
properties resulting from the binding of PSA (Gharatape, et al., 2019).
 Piezoelectric Biosensors: Piezoelectric transducers detect changes in mass or
viscosity upon PSA binding, leading to alterations in the resonant frequency of the
device (Pohanka, et al., 2018).
 Calorimetric Biosensors: Calorimetric biosensors measure the heat generated or
absorbed during the binding of PSA, enabling its quantification (Sayed, et al., 2019).

High sensitivity, specificity, and the ability for real-time analysis are just a few benefits that
biosensors for PSA detection offer. These biosensing technologies make it possible to detect

15
PSA in biological matrices like blood, urine, or saliva quickly and accurately. Biosensors aid in
the early diagnosis, treatment, and patient outcomes of prostate cancer by enabling the
monitoring and early detection of PSA levels.
2.3 Types of Fabricated Electrodes
For PSA (Prostate-Specific Antigen) detection, the choice of electrode material is crucial to
ensure high sensitivity and selectivity. Carbon-based electrodes, specifically modified with
biomaterials or nanomaterials, are commonly used for PSA detection. Here are some electrode
options suitable for PSA detection:

 Carbon nanotube electrodes: Carbon nanotubes (CNTs) offer excellent electrical

conductivity and a high surface-to-volume ratio, making them suitable for biosensing
applications. Functionalizing CNT electrodes with antibodies or aptamers specific to PSA
can enable selective detection.

Carbon nanotube (CNT) electrodes are a cutting-edge development in materials science

and nanotechnology. These unusual structures, made of rolled-up graphene sheets, are
widely sought-after for a variety of applications in electronics, energy storage, and
biosensing due to their extraordinary features.
One of the most important properties of carbon nanotubes is their excellent electrical
conductivity. Since they have great electron mobility, charge may be transported through
their cylindric structure effectively. They are the perfect electrode materials for batteries,
supercapacitors, and solar cells thanks to this characteristic. By increasing energy storage
capacity, power density, and charge/discharge rates, CNT electrodes can improve these
devices' overall performance.
Carbon nanotubes have great mechanical strength and flexibility in addition to their
exceptional electrical capabilities. They are incredibly strong and deformation-resistant
thanks to their distinctive structure, which can sustain mechanical stress and strain. For
applications where electrodes must withstand challenging circumstances or frequent
mechanical deformation, such as wearable electronics or flexible displays, this
mechanical robustness is especially helpful.
The large surface area of carbon nanotube electrodes is another major benefit. Numerous
active sites in CNTs make it possible for electrochemical reactions to occur. The huge

16
 surface area of CNT electrodes enables effective charge storage and quick ion movement,
making them particularly useful in energy storage systems like supercapacitors.
Additionally, it makes CNT electrodes more capable of adsorbing biomolecules, which
makes them ideal for biosensing applications.
Additionally, a variety of chemical groups can be added to carbon nanotubes to provide
them with new functions and improve their properties. By affixing molecules or
nanoparticles to the CNT surface, functionalization can be accomplished, permitting
tailored interactions with substances or encouraging particular chemical reactions. This
feature offers fascinating opportunities for tailoring CNT electrodes for uses like
selective sensing or catalysis.
Additionally, improvements in neural interfaces and bioelectrodes have been made
possible by the invention of carbon nanotube electrodes. They are ideal for interacting
with biological systems because of their distinctive combination of electrical
conductivity, mechanical flexibility, and biocompatibility. Researchers can better
comprehend the complexity of the brain and create new treatments for neurological
illnesses by recording and stimulating neural activity with CNT electrodes.
Carbon nanotube electrodes have a lot of potential, but there are still several problems
that need to be solved. A considerable challenge still exists in the large-scale manufacture
of high-quality CNTs at a reasonable price. Further research is required to address
concerns with CNT alignment, homogeneity, and controlled development to guarantee
consistent and dependable electrode performance.
In conclusion, carbon nanotube electrodes are a ground-breaking technology with a wide
range of uses in neural interfaces, energy storage, electronics, and biosensing. They are
highly sought-after for a variety of developing technologies due to their superior
electrical conductivity, mechanical strength, vast surface area, and functionality. For
carbon nanotube electrodes to reach their full potential and assist a variety of industries,
more research and development in this area is essential.

 Graphene-based electrodes: Graphene, a two-dimensional carbon material, exhibits

excellent electrical conductivity and a large surface area. Graphene electrodes modified
with PSA-specific antibodies or aptamers can facilitate sensitive detection of PSA.

17
Medical diagnostics have advanced significantly in recent years, especially with the
incorporation of electrodes constructed of graphene. These electrodes have become a
cutting-edge technique for the early and precise diagnosis of prostate cancer by utilizing
the extraordinary features of graphene. In this article, we examine how graphene-based
electrodes are transforming the diagnosis of prostate cancer by providing unprecedented
precision and sensitivity.
The most popular protein indicators for the identification of Prostate Cancer:
Typically, cancer cells or other cells in response to the protein biomarkers for cancer
diagnosis to cancer (Li Xu, et al., 2019), which have proven to be effective targets for
early detection, tracking therapy effectiveness, spotting recurrence, or keeping track of
prognosis tumors (Li Xu, et al., 2019). Protein biomarkers are typically unstable and low
in abundance in bodily fluids, making it difficult to specifically identify them in these
environments (Li Xu, et al., 2019). Basic needs to be considered while fabricating protein
biosensors include sensitivity, specifity, and accuracy (Li Xu, et al., 2019).
One of the earliest discovered serological PC indicators, prostate-specific antigen (PSA)
(Stamey, et al., 1993), also known as human kallikrein 3 (hK3 or KLK3), has received
widespread recognition in clinical use (Li Xu, et al., 2019). PSA levels above 4.0 ng/mL
are typically regarded as abnormal (Liu, et al., 2013), making this value the
internationally accepted cutoff point for PC incidence (Li Xu, et al., 2019). Since greater
PSA levels can be detected in benign illnesses such benign prostatic hyperplasia (BPH
(Li Xu, et al., 2019), the specificity of PSA is still limited (Perez-Ibave, et al., 2018).
Additionally, PSA can be produced by both healthy breast cells and breast cancer cells.
These restrictions suggest that using PSA as a sole surrogate marker for PC diagnosis and
screening is inappropriate. Thankfully, numerous other protein PC indicators have been
created.
A type II transmembrane protein called prostate-specific membrane antigen (PSMA)
(Israeli, et al.,1993) has been shown to be expressed in benign prostatic hyperplasia and
at higher levels in prostatic adenocarcinoma and high-grade prostatic intraepithelial
neoplasia (Bostwick, et al.,1998).
Additionally, higher PSMA expression is associated with malignancy (Li Xu, et al.,
2019). The results of the available study point to a potential clinical application for

18
PSMA in PC patients. Therapeutics and imaging have been the main PSMA clinical
applications so far (Li Xu, et al., 2019). Another recently identified PC biomarker is
prostate stem cell antigen (Reiter, et al., 2013), which is hardly found in normal tissues
but is abundantly expressed by several types of human prostate cancers, including
metastatic and hormone-refractory tumors (Li Xu, et al., 2019). Prostatic cancer patients'
urine samples include the Engrailed-2 (EN2) protein, which has a specificity of 88.2%
and a sensitivity of 66% (Li Xu, et al., 2019). Therefore, it is well acknowledged that
EN2 in urine may be a biomarker of PC.

Properties of graphene materials for biosensors

Two-dimensional (2D) nanomaterial graphene has a significant impact on the biosensor
field (Li Xu, et al., 2019). Amazing physical, optical, electrochemical, and magnetic
capabilities are provided by the application of graphene in biosensing platforms (Li Xu,
et al., 2019). Research on various types of graphene materials, such as pure graphene and
functionalized graphene such as graphene oxide (GO), reduced graphene oxide (rGO),
and graphene-based quantum dots (GQDs), etc., is ongoing in the field of biosensors (Li
Xu, et al., 2019). The array of a 2D hexagonal lattice composed of sp2 -bonded carbon
atoms is what is known as pure graphene. By chemically oxidizing and peeling off
graphene, GO is created, significantly altering the basal plane in the process (Li Xu, et
al., 2019). Te rGO is created by reducing GO, and many techniques have been discovered
to do this, including thermal, chemical, microwave, photochemical, microbial/bacterial,
and photo-thermal processes (Li Xu, et al., 2019). GQDs, which exhibit quantum
phenomena, are made up of single to tens of layers of graphene with a thickness of a few
nanometers (Li Xu, et al., 2019).
First, to create novel biosensor interfaces for enhanced molecular receptor assembly,
functionalized graphene materials such as GO, rGO, and GQDs (Suvarnaphaet, et al.,
2017) were assembled onto the biosensor surface [electrode, feld-effect transistors (FET)
channel, etc.]. Excellent biosensor performance in this group was primarily made
possible by the enhanced specific surface area and the distinctive - orbital interaction on
the interface. Second, a lot of recent research has utilized graphene materials as superior
carriers for the creation of innovative nanocomposites (Li, et al., 2018). In this group,

19
intriguing biosensor signal amplification and distinctive catalytic/chemical activity were
achieved for sensitive protein biomarker analysis (Li, et al., 2016).
Construction of antibody-graphene biosensor interface
Traditional immune-assay surfaces, such as conventional 96-well plates and colloidal
gold test strips, allow antibodies to be physically absorbed. The ability and capacity,
however, is one of the key challenges since the hydrophobic and hydrophilic connection
is comparatively weak and the antibody molecules' orientation is random (Sakhnini Li et
al., 2016). According to several recent research, the strong cross-linking between the
amine groups of antibodies and the carboxylic acid groups on graphene materials
(COOH-NH2) was employed to assemble antibodies on new biosensor interfaces (Li Xu,
et al., 2019). The use of graphene materials in their research improved the loading
quantity, orientation controllability, and binding capacity of the antibodies or antibody
fragments. For example, Li, et al., 2016 developed a graphene modified sensor platform
with increased surface area, and then assembled antibody onto the surface through
COOH–NH2 combining, with the assistant of 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and they finally achieved a low
detection limit of 2 pg/mL (Li, et al., 2011).
Mao, et al., used chitosan as the dispersant to create an immuno-interface on a glassy
carbon electrode, which supplied much more amino groups for PSA antibody attachment
and enabled better-oriented antibody assembly. They finally created a straightforward,
label-free electrochemical immunosensor using an electrode modified with graphene-
methylene blue composite (Mao, et al., 2012). A unique 3D graphene-Au composite
created more recently by Jang, et al., has a larger accessible surface area for antibody
combination than a 2D graphene sheet. More significantly, greater capacitances may be
produced by crumpled graphene, which is essential for the subsequent electrochemical
immunosensing (Jang, et al., 2015). A graphene-modified electrode for PSA detection
was also described in an ECL biosensor (Xu, et al., 2011). Recently, an electrode surface
modified with Au/Ag-rGO was created by Wu, et al. The electrode surface was
subsequently mixed with a significant number of aminated GQDs and carboxyl GQDs. In
their research, PSA antibody was adsorbed onto Au and Ag nanoparticles, and GQDs

20
were employed to amplify the ECL signal. Finally, they created a label-free PSA ECL
biosensor with a 0.29 pg/mL detection limit (Wu, et al., 2016).

Fig 1. Schematic illustration of Label-free electrochemical immunosensors for PC protein biomarkers

based on: A graphene-methylene blue nanocomposite, Reprinted with permission from [93], Copyright
2011 Elsevier. B graphene-Au nanocomposite (Reprinted with permission from [94]. Copyright 2014
Elsevier).

In addition, graphene materials were used to build 2D nano-FET biosensors, which have
unique benefits over 1D FET biosensors in terms of having more receptor biomolecules,
low noise, and high sensitivity (Li Xu, et al., 2019). An effective example is the label-
free, ultrasensitive rGO-based FET biosensor (Kim, et al., 2013) for the analysis of
PSA/1-antichymotrypsin (PSA-ACT). By integrating rGO onto an aminated glass surface
and functionalizing it with PSA antibody, the FET biosensor was created. The gate

21
 voltage (Vg,min) shifted linearly when PSA-ACT was bound by the antibodies on the
FET substrate, demonstrating minimal conductivity. Finally, they were able to detect
PSA-ACT at a femtomolar level.

 Conducting polymer electrodes: Conducting polymers, such as polyaniline or

polypyrrole, can be used as electrode materials for PSA detection. These polymers can be
electrochemically synthesized or modified with biomolecules to enhance the detection
sensitivity and selectivity.
 Gold electrodes: gold electrodes functionalized with thiolated antibodies or aptamers
specific to PSA can be employed for PSA detection using electrochemical or surface
plasmon resonance (SPR) techniques.
 Silicon-based electrodes: Silicon-based electrodes, such as silicon nanowires or silicon
nanowire field-effect transistors (SiNW-FETs), have shown promise for sensitive PSA
detection. The surface of these electrodes can be functionalized with PSA-specific
biomolecules to enable label-free detection.

2.4 Immobilization Strategies for PSA Detection

Using immobilization methodologies, biomolecules like antibodies, enzymes, aptamers, or
receptors can be fixed or attached to a stable surface or support. By interacting with target
analytes or antigens, the biomolecules can facilitate the detection, quantification, or other
biological interactions that are sought.
Immobilization is frequently done to improve the stability, specificity, and sensitivity of
numerous biotechnological applications, biosensors, and bioanalytical assays. Biomolecules can
be immobilized to maintain their activity and selectivity and to prevent them from being lost or
washed away during sample processing.
When it comes to detecting prostate-specific antigen (PSA), immobilization strategies play a
crucial role in improving the sensitivity and selectivity of detection methods. Here are a few
commonly used immobilization strategies for PSA detection:

 Antibody immobilization: Immobilizing antibodies specific to PSA onto a solid surface is

a widely used approach. Antibodies can be immobilized using physical adsorption,
covalent binding, or biotin-streptavidin interactions. This allows the antigen-antibody
interaction to occur, facilitating the detection of PSA.

22
Immunosensing, a highly selective analytical technique that makes use of antibodies'
ability to recognize molecules, is greatly aided by antibody immobilization (Shinji
Sueda1, 2022). Due to its great specificity and sensitivity, immunosensing has found use
in a variety of sectors, including medical diagnosis, food analysis, and environmental
monitoring (Shinji Sueda1, 2022).
In immunosensing, antibodies are frequently immobilized on solid supports of sensing
platforms to monitor the signals produced by the binding of antibodies to antigens to
detect antigens. Traditional immobilization techniques, such physical adsorption or
covalent coupling, can cause denaturation, unpredictable orientation, and steric hindrance
in antibodies on solid substrates, which reduces their capacity to bind antigens
(Makaraviciute, et al., 2013). Numerous strategies for the oriented immobilization of
antibodies have been devised to address these issues.
Using antibody-binding proteins like Protein A and Protein G, which are produced from
staphylococcal and streptococcal bacteria, respectively, is one efficient method for
antibody immobilization. These proteins bind to antibodies' Fc regions with specificity,
but they have no effect on how well they can bind antigens. Antibodies can be site-
specifically captured by immobilizing Fc-binding proteins bearing tag sequences on the
sensing substrates, leaving the Fab region completely open for antigen detection (Shinji
Sueda1, 2022).
For instance, Fc-binding proteins with numerous cysteine residues or a gold-binding
peptide have been successfully mounted on sensor chip gold surfaces to successfully
capture antibodies (Shinji Sueda1, 2022). Like this, the synthetic IgG-binding domain (Z-
domain) of Protein A carrying a His-tag was immobilized on iminodiacetic acid-Ni2+
complex-coated microtiter plates, and the IgG-binding domains of Protein G fused to
glutathione S-transferase were immobilized on glutathione-modified gold surfaces (Shinji
Sueda1, 2022). In each of these situations, oriented antibody immobilization led to
improved immunosensing performance compared to systems with randomly oriented
antibodies.
To provide optimal sensitivity, a high surface density of antibodies on solid supports
must be achieved during the antibody immobilization process. Methods utilizing highly
ordered molecular structures have been suggested as a solution to this. For instance,

23
 surface-initiated radical processes have been used to produce polymer chains on solid
substrates for detection systems using polymer brushes, and antibodies have been added
to the various binding sites within each polymer chain. It has been used to detect several
diagnostic marker proteins and improves antibody-loading capability. It is also possible
to reduce non-specific protein absorption by including hydrophilic functional groups in
the polymer chains (Shinji Sueda1, 2022).
The use of poly(amidoamine) dendrimers immobilized on self-assembled monolayers,
bionanocapsules incorporating the Z-domain of Protein A, and protein assemblies
containing enzymes, substrate proteins, and the Z-domain of Protein A are additional
methods with highly organized molecular architectures. These methods have special
qualities that make them suited for immunosensing (Shinji Sueda1, 2022).

 Self-assembled monolayers (SAMs): SAMs are formed by the spontaneous adsorption of

molecules onto a substrate, creating a thin, organized layer. Functionalized SAMs can be
used to immobilize antibodies or peptides that selectively bind to PSA. SAMs provide a
stable and well-defined surface for detection.
 Aptamer immobilization: Aptamers are single-stranded DNA or RNA molecules that can
bind to specific target molecules with high affinity and selectivity. Immobilizing PSA-
specific aptamers onto a surface allows for the capture and detection of PSA in a
sensitive manner.

The construction of electrochemical aptasensors requires the use of aptamer

immobilization techniques since they have a substantial impact on the sensor's overall
performance (Meirinho et al., 2016). A bioreceptor, such as an aptamer, is attached to or
conjugated to a transducer to become immobilized (Eggins et al., 2004). The choice of
immobilization method is essential for preserving the aptamer's stability, affinity, and
specificity toward the target analyte. The electrode surface, bioreceptor characteristics,
target analyte physicochemical characteristics, and biosensor fabrication conditions are
some of the variables that affect the preferred immobilization approach. To successfully
immobilize as a bioreceptor, the chosen immobilization method must guarantee the
maintenance of aptamer affinity and selectivity (Meirinho et al., 2016).

24
Physical adsorption and covalent bonding approaches are the most often utilized methods
for aptamer immobilization (Meirinho et al., 2016). These techniques include self-
assembled monolayers (SAM) with thiol-based interactions, streptavidin-biotin
interactions, and surface activation with EDC/NHS. The immobilization method used
affects the aptamer's surface coverage, which in turn affects how well the aptamer binds
to the target analyte (Jarczewska et al., 2014).
In physical adsorption-based immobilization, aptamer modification is not necessary;
instead, electrostatic forces are used to immobilize the probe onto the electrode surface.
The desorption of the aptamer from the electrode surface results in low stability, which
makes this approach, despite its simplicity, unsuitable for the construction of aptasensors
(Meirinho, et al., 2016).
The detection of the HIV-1 gene (Gong, et al., 2017) is one instance of an
electrochemical aptasensor created via physical adsorption. This aptasensor used
electrochemical impedance spectroscopy (EIS) for detection and a modified glassy
carbon electrode (GCE) made of a graphene-Nafion composite. The graphene-Nafion-
modified GCE surface was adsorbed with the single-stranded DNA (ssDNA) probe. The
target analyte's DNA and the single-stranded DNA probe interacted to generate double-
stranded DNA (dsDNA), which dissociated from the aptasensor surface because it
interacted with graphene less strongly than ssDNA and complementary DNA did (Gong,
et al., 2017).
For immobilization on the electrode surface, aptamers can also be altered with functional
groups like thiols (-SH), biotin, and amino (-NH2) groups. Thiol-based interactions are
frequently used in self-assembled monolayers (SAM), which rely on the strong affinity
between thiol groups (-SH) and gold surfaces. Covalent bonds between sulfur and gold
can develop because of this interaction (Arum, et al., 2022).
Cyclic voltammetry (CV) was employed by Mohsin, et al., to identify the Hepatitis B
virus in an electrochemical aptasensor. Graphene oxide-functionalized gold nanoparticles
(AuNPs) were used to modify the GCE's surface. The Hepatitis B surface antigen
(HBsAg)-recognizing aptamer, modified with thiol groups (-SH), was covalently bound
on the modified GCE surface using AuNPs via significant gold-sulfur affinity. As a redox
probe, methylene blue (MB) was used, and it was electrostatically bound to the guanine

25
 base and intercalated into the aptamer structure. The precise binding between the aptamer
and the HBsAg target analyte was the basis for the aptasensor's operation. There was a
strong electrochemical signal produced when the target analyte wasn't present but when
the target analyte was present, the target analyte's presence triggered the aptamer to
dissociate, which led to the release of MB from the aptas.

 Molecular imprinting: Molecularly imprinted polymers (MIPs) can be used to create

artificial recognition sites that mimic antibodies or aptamers. MIPs are synthesized by
polymerizing monomers in the presence of a template molecule (such as PSA) and
subsequently removing the template. The resulting polymer matrix contains specific
binding sites for PSA, enabling its immobilization and subsequent detection.

MIPs are polymeric matrices with distributed binding cavities designed to match the
imprinted template's dimensions, form, and functionality. Figure 2 as stated by T. Cowen,
et al., 2016) shows the typical course of events that lead to the creation of MIPs:

Fig 2. Schematic of the general production methodology for a Molecularly Imprinted Polymer. A pre-
polymerization mixture allows for the non-covalent interactions to stabilize between the template and the
functional monomers. The polymerization process then freezes the template inside the polymer matrix. The
template is then removed from the polymer leaving a binding cavity of complementary size, shape and
functionality to the target. When the target is introduced, it will bind back inside the cavity.

In 1949, Dickey released the first English study on molecularly imprinted polymers
(MIPs), introducing crucial ideas for MIP development, such as the designation of the

26
target molecule as a "template" (Dickey, et al., 1949). Mosbach's group developed non-
covalent imprinting in the 1980s, which is now widely employed in research (Crapnell et
al., 2020).

1. Incubation: Monomers are incubated with a template molecule, dummy template,

or epitope, allowing for non-covalent interactions between the functional
monomers and the template, leading to their stabilization.
2. Polymerization: The monomers undergo polymerization, forming a polymer
around the template, effectively trapping it within the polymeric matrix.
3. Template Removal: The template is removed or extracted from the polymer,
leaving behind specific binding cavities that match the size and shape of the
template.

Before synthesis, interactions between monomers and templates can be studied using
computational modeling, which can help with technique selection (Cowen, et al., 2020).
There are numerous synthetic methods that can be used to create MIP, including UV
polymerization, thermal polymerization, RAFT polymerization, and solid-phase synthesis
(Crapnell, et al., 2020). The qualities and morphologies of the finished MIP product may
differ significantly because of these operations. Due to reasons such restricted mass
transfer, permanent entrapment in the polymer matrix, poor structural integrity,
constrained synthetic pathways, solvent choice, and heterogeneity of binding sites,
traditional bulk imprinting has proven difficult when imprinting proteins (Crapnell, et al.,
2020).
Electropolymerization is frequently selected as the method for MIP synthesis when MIPs
are combined with electrochemical detection (Crapnell, et al., 2020). Depending on the
required platform and electrochemical detection technique, this procedure can produce
conductive or non-conductive polymers, which are compatible with the overall platform.
Additionally, electropolymerization makes it possible for the MIP layer to be formed
directly on the transducer surface, doing away with the requirement for laborious
immobilization processes. Multiple biological targets have been successfully detected
using electropolymerization in MIP creation (Crapnell, et al., 2020). However, issues like

27
 the scarcity of appropriate monomers, the homogeneity of binding sites, and the process'
scalability must be resolved.
Alternative synthesis techniques such as dip-coating, screen-printing, and sol-gel
techniques have resulted in MIPs that perform better. The reproducibility of
immobilization techniques or integration with the read-out approach, however, may
provide challenges for these technologies (Crapnell, et al., 2020).
The synthesis, template removal, and detection parameters need to be carefully optimized
to reach the essential levels of detection and reliability for successful sensor devices
targeting clinical biomarkers.
Recent reports of electrochemical MIP sensors for clinically important biomarkers are
emphasized throughout the paper, along with discussions of their successes and
opportunities for development.

 Nanomaterial-based immobilization: Various nanomaterials, such as gold nanoparticles,

magnetic nanoparticles, carbon nanotubes, and graphene oxide, can be functionalized and
used for immobilizing PSA-specific ligands. These nanomaterials offer enhanced surface
area, biocompatibility, and improved signal amplification for sensitive detection.

Enzyme immobilization, a technique discovered in 1916 (Nelson, et al.,1916) involves

the adsorption of enzymes onto solid matrices, such as charcoal or aluminum hydroxide,
without compromising their activity. This breakthrough paved the way for the
development of various enzyme immobilization techniques that are widely used today
(Nelson, et al., 1916).
Initial enzyme loadings in comparison to accessible surface areas were modest for
enzyme immobilization approaches. However, several covalent techniques for
immobilizing enzymes were created in the late 1990s, which led to improved efficiency
and commercial uses of immobilized enzymes (Razi Ahmad, et al., 2015). The
immobilization of enzymes on solid substrates has improved their stability and handling
by increasing their resilience to environmental changes like pH and temperature (Cherry,
et al., 2003). Because the reaction products are free of the enzyme itself, immobilized
enzymes are extremely valuable in sectors like food and medicines (Cherry, et al., 2003).

28
The decrease of autolysis processes, particularly in proteases, brought about by
multipoint or multiunit immobilization or by fostering a suitable enzyme environment is a
key benefit of enzyme immobilization (Massolini, et al., 2005). Additionally,
immobilization enhances several enzyme characteristics, such as selectivity, thermal
stability, performance in organic solvents, and pH tolerance. Immobilization imparts
structural stiffness that inhibits dissociation-related inactivation and makes the associated
enzyme readily accessible for subsequent reactions without requiring additional
extraction and purification steps (Guzik, et al., 2014).
The structural modifications made during the immobilization procedure and the
development of a microenvironment distinct from the bulk solution are what lead to these
improvements in enzyme characteristics (Rodrigues, et al., 2013). By using a support
material with a cheap manufacturing cost and high binding capacity, enzyme
immobilization's primary goal is to optimize the benefits of enzyme catalysis (Secundo,
et al., 2013).
The characteristics of the interaction between the enzyme and the carrier, the number and
positions of bonds formed, the conformational freedom in the matrix, the
microenvironment around the enzyme, the chemical and physical characteristics of the
carrier, the characteristics of the spacer connecting the enzyme molecules to the carrier,
and the immobilization conditions are all factors that affect the stability of immobilized
enzymes (Mateo, et al., 2007). As a result, depending on factors including time,
temperature, storage conditions, and experimental variables, the stability of immobilized
enzymes might either increase or decrease (Mateo, et al., 2007).
Enzyme immobilization can be achieved through physical or chemical methods. Physical
methods involve weak interactions between the matrix and the enzyme, while chemical
methods involve the formation of covalent bonds between the support and the enzyme.
Recent advancements in organic chemistry and molecular biology have led to the
development of powerful and efficient site-specific protein immobilization techniques,
enabling important applications such as functional protein microarrays, biosensors, and
continuous flow reactor systems (Ahmad, et al., 2015).
In summary, enzyme immobilization techniques have revolutionized enzyme applications
by increasing stability, improving properties, and enabling their use in various industries.

29
The choice of immobilization method, whether physical or chemical, depends on the
specific requirements of the application. Ongoing research in this field reflects the
continued interest and the potential for further advancements in enzyme immobilization.

2.5 Nanomaterials as Immobilization Matrix

Due to their excellent properties that perfectly balance the important elements affecting
the effectiveness of biocatalysts, nanoparticles have become highly effective support
materials for enzyme immobilization. These variables include effective enzyme loading,
specific surface area, and mass transfer resistance (Ahmad, et al., 2015). Diffusion
becomes a bigger problem when working with macromolecular substrates, and
nanoparticles are the perfect solution (Hwan, et al., 2013). Additionally, when
disseminated in aqueous solutions, enzymes immobilized on nanoparticles show
Brownian mobility, indicating that their enzymatic activity are substantially superior to
those of unbound enzymes (Gupta, et al., 2011).
Particularly advantageous are magnetic nanoparticles since they may be easily separated
using an external magnetic field. Enzyme immobilization on nanoparticles has been
found to decrease protein unfolding and enhance stability and performance (Gupta, et al.,
2011). The immobilization of enzymes on various kinds of nanoparticles, such as metal
nanoparticles, metal oxide nanoparticles, magnetic nanoparticles, porous nanoparticles,
and polymeric nanoparticles, has been the subject of several reviews (Ahmad, et al.,
2015).
There have been numerous reports of enzymes that have been nano immobilized.
Examples include the immobilization of lysozyme, glucose oxidase, aminopeptidase, and
alcohol dehydrogenase on gold and silver nanoparticles (Ahmad, et al., 2015). For use in
non-aqueous medium, enzymes such Candida antarctica lipase B (CALB) and S.
Carlsberg have been immobilized on fumed silica nanoparticles and demonstrate very
high catalytic activity (Cruz, et al., 2011). Acetylcholinesterase has been immobilized for
paraoxon detection using magnetic glasses based on iron oxide/silica (Won, et al., 2010).
Additionally, acetylcholinesterase has been immobilized on nickel nanoparticles, offering
a highly sensitive method of pesticide detection that uses organophosphates (Ganesana,
et al., 2011). -amylase has been immobilized using magnetic poly (2-hydroxyethyl

30
methacrylate-N-methacryloyl-(l)-phenylalanine), which still exhibits 85% specific
activity after 10 re-uses (Uygun, et al., 2012). At 80 °C, cellulase bound on magnetic
nanoparticles shows marginally higher activity than the free enzyme as stated by
(Khoshnevisan, et al., 2011). Trypsin has been immobilized using amino-functionalized
silica-coated magnetic nanoparticles for pressure-assisted digestion in proteome analysis,
yielding more protein identifications than free trypsin (Lee, et al., 2011). By employing
aminofunctionalized Fe3O4SiO2 nanoparticles covalently bound to ferrocene
monocarboxylic acid as the building block for glucose biosensors, it has been possible to
achieve a steady-state current of 95% within 10 seconds of the injection of glucose (Qiu,
et al., 2007).
Recent studies have successfully immobilized enzymes like peroxidase, cellulase, trypsin,
and alpha amylase on TiO2 nanoparticles. The immobilized enzymes have shown
increased thermal stability at higher temperatures as well as higher activity when
compared to free enzymes (Ahmad, et al., 2015).
When nanomaterials are utilized as solid supports, the benefits of immobilized enzymes
on micron-sized particles are also applicable. Electrostatic adsorption, covalent
attachment to surface-modified nanoparticles, conjugation using a protein's unique
affinity, and direct conjugation to the nanoparticle surface are the four basic methods
used to attach proteins or enzymes to nanoparticles (Ahmad, et al., 2015). The most
popular technique, electrostatic adsorption, includes a straightforward interaction
between the nanoparticle and the protein that may be controlled by pH or charge
screening (Geoghegan, et al., 1977). Through surface chemistry control, covalent
attachment, which entails the covalent binding of a protein to the nanoparticle ligand, can
be accomplished (Aubin-Tam, et al., 2008). For nanoparticle-protein conjugation,
specialized labeling techniques can also be used, such as streptavidin-coated
nanoparticles that selectively bind biotin-labeled proteins or antibody-coated
nanoparticles that specifically bind proteins (Di Marco, et al., 2010). For example, the
Au-thiol or Ag-thiol chemistry for gold or silver nanoparticles immediately reacts with
the surface of the protein in direct conjugation (Aubin-Tam, et al., 2008).
For nanoparticle-protein conjugation, specialized labeling techniques can also be used,
such as streptavidin-coated nanoparticles that selectively bind biotin-labeled proteins or

31
 antibody-coated nanoparticles that specifically bind proteins (Di Marco, et al., 2010). For
example, the Au-thiol or Ag-thiol chemistry for gold or silver nanoparticles immediately
reacts with the surface of the protein in direct conjugation (Aubin-Tam, et al., 2008).
In conclusion, because of their beneficial properties, nanoparticles make an excellent
support material for the immobilization of enzymes. High specific surface area, efficient
mass transfer, and enhanced enzyme loading are all provided. Compared to their free
counterparts, enzymes immobilized on nanoparticles exhibit increased enzymatic activity,
stability, and performance. Applications in numerous disciplines, including biosensing,
BioCatalysis, and proteome analysis, have resulted from the effective use of a variety of
nanoparticle types, including magnetic nanoparticles, for enzyme immobilization. Future
studies should concentrate on creating scalable, affordable synthesis processes for
nanoparticles to enable their wider application in enzyme immobilization and other fields.

 Microfluidic immobilization: Microfluidic platforms can be utilized for PSA detection by

immobilizing antibodies, aptamers, or MIPs within microchannels or on microbeads.
These platforms allow for efficient capture and separation of PSA, enabling rapid and
sensitive detection.

The concepts of microfluidics and immunoassays are combined in a cutting-edge method

called microfluidic immobilization for prostate-specific antigen (PSA) detection, which
enables quick, sensitive, and precise evaluation of PSA levels in clinical samples. The
prostate gland produces a protein called PSA, which is frequently utilized as a biomarker
for the early detection and follow-up of prostate cancer.
Enzyme-linked immunosorbent assays (ELISA), a common traditional approach for PSA
detection, frequently call for large sample volumes, protracted processing times, and
specialized laboratory apparatus. These restrictions are solved by microfluidic
immobilization, which integrates numerous analytical procedures into a single device and
miniaturizes the detection process.
Typically, a microchannel network with carefully considered shapes and functionalized
surfaces makes up the microfluidic immobilization platform. Specific antibodies or
antigen-binding substances are added to these surfaces so that they can catch PSA
molecules from the sample solution with precision. The concentration of the target

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molecules and subsequent, more sensitive detection are made possible by their
immobilization.
The microfluidic immobilization process for PSA detection has multiple phases. The
clinical sample is first introduced into the microchannel network, such as blood or serum.
The immobilized antibodies or capture agents on the channel surfaces are in contact with
the sample as it passes through the channels, where the PSA molecules in the sample bind
to them. This process is essential for isolating PSA from other components in the sample
and minimizing interference.
The microfluidic device is cleaned to get rid of impurities and unattached molecules after
the immobilization stage. This washing process contributes to improving the assay's
selectivity and lowering background noise. After washing, the microchannels are then
filled with a detection reagent, usually a tagged antibody or probe. A sandwich-like
compound is created when the detecting reagent binds to the immobilized PSA
molecules.
The measurement or quantification of the engulfed PSA molecules is the last stage of
microfluidic immobilization. This is accomplished using a variety of detection
techniques, such as electrochemical sensing, fluorescence, chemiluminescence, or
colorimetry. The exact design of the microfluidic device and the instrumentation at hand
influence the choice of detection technique.
Microfluidic immobilization for PSA detection has a few benefits, one of which is its
capacity for quick and accurate results. The analysis of small sample volumes is made
possible by the compact design of microfluidic devices, which also speeds up assays and
uses less chemicals. Additionally, the constrained microscale environment improves the
kinetics of mass transfer and interaction, resulting in higher sensitivity and lower
detection limits.
Moreover, point-of-care testing and dispersed diagnostics are possibilities using
microfluidic immobilization. Microfluidic devices are suited for usage in settings with
limited resources or at the patient's bedside due to their tiny size, portability, and
simplicity. This can facilitate early prostate cancer identification, speed up the diagnostic
procedure, and provide real-time monitoring of PSA levels throughout therapy.

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To sum up, microfluidic immobilization for PSA detection is a promising strategy that
combines the benefits of immunoassays and microfluidics to offer quick, accurate, and
portable diagnostic capabilities. By enabling affordable and accessible testing procedures,
this technology has the potential to change the field of prostate cancer screening and
monitoring. Future advancements in this field of study and development are anticipated
to substantially enhance the functionality and application of microfluidic immobilization
for PSA detection.

2.6 Signal Transduction methods used in PSA Detection.

The measurement of PSA levels in the blood is a crucial step in the diagnosis and
surveillance of prostate cancer. The prostate gland's cells create a protein called PSA, and
both prostate cancer and other non-cancerous illnesses that affect the prostate can cause
increased PSA levels.
The most popular technique for detecting PSA is immunoassays, which depend on
antibodies specifically attaching to PSA molecules. There are numerous immunoassay
methods available for the detection of PSA, some of which are frequently utilized.
1. Enzyme-Linked Immunosorbent Assay (ELISA): This technique uses enzyme-labeled
antibodies to detect the presence of PSA. The reaction produces a colored product, and
the intensity of the color is directly proportional to the concentration of PSA in the
sample.
2. Chemiluminescent Immunoassay (CLIA): In CLIA, antibodies are labeled with a
chemiluminescent substance. When the labeled antibodies bind to PSA, the
chemiluminescent reaction occurs, producing light. The light emission is measured and
correlates with the amount of PSA present in the sample.
3. Fluorescence Immunoassay: This method uses fluorescent-labeled antibodies to detect
PSA. When the labeled antibodies bind to PSA, fluorescence is emitted, and the intensity
of the fluorescence is proportional to the PSA concentration.
4. Radioimmunoassay (RIA): In RIA, radioactive isotopes are used to label the
antibodies. The binding of the labeled antibodies to PSA is measured by the level of
radioactivity, and this is used to determine the PSA concentration.

34
5. Immunofluorescence: In this technique, antibodies labeled with fluorescent molecules
are used to detect PSA. When the labeled antibodies bind to PSA in the sample, they emit
fluorescence, which is then visualized and quantified using a fluorescence microscope or
a specialized instrument.

2.7 Performance optimization and enhancement techniques

 Advanced PSA Testing Technologies:

Through the provision of data beyond the conventional total PSA measurement, advanced
PSA testing devices seek to increase the accuracy and reliability of prostate cancer
detection. The following are some of the main cutting-edge PSA testing technologies:
Free PSA (fPSA): Prostate-Specific Antigen circulates in the blood in two major forms:
complexed PSA, which is PSA that hasn't been coupled to any other proteins, and free
PSA. Prostate cancer and benign diseases of the prostate can be distinguished from one
another using the fPSA ratio, which measures the proportion of free PSA to total PSA. In
general, a higher risk of prostate cancer is linked to a smaller percentage of free PSA.
The fPSA ratio is determined by measuring the levels of both free PSA (fPSA) and total
PSA (tPSA) and then calculating the percentage of free PSA in relation to the total PSA.
The formula for calculating the fPSA ratio is as follows:
fPSA Ratio = (Free PSA / Total PSA) × 100
In this formula:
Free PSA refers to the amount of unbound PSA in the blood.
Total PSA refers to the sum of both free PSA and complexed PSA.
The fPSA ratio is expressed as a percentage. Higher fPSA ratios indicate a higher
proportion of unbound (free) PSA relative to the total PSA. A higher percentage of free
PSA is generally associated with a lower risk of prostate cancer. Conversely, a lower
fPSA ratio (a lower percentage of free PSA) may suggest a higher likelihood of prostate
cancer.

35
The fPSA ratio is one of the parameters used in conjunction with other clinical
information, such as age, family history, digital rectal examination (DRE) findings, and
other risk factors, to help determine the need for further evaluation or a prostate biopsy.
It's important to note that while the fPSA ratio can provide valuable information, it is not
a definitive diagnostic test for prostate cancer. A prostate biopsy is still necessary to
confirm the presence of cancer cells in the prostate tissue. As with any medical test, the
interpretation of fPSA ratio results should be discussed with a qualified healthcare
professional.
PSA Velocity: The rate at which PSA values change over time is referred to as PSA
velocity. Prostate cancer risk may increase if PSA levels significantly rise over a short
period of time. To effectively evaluate PSA velocity, it is crucial to consider several
additional variables, including age and baseline PSA levels.
To calculate PSA velocity, follow these steps:

 Choose at least two PSA test results taken at different times. The time interval
between the tests should ideally be at least 12 months.
 Subtract the initial PSA value from the later PSA value.
 Divide the difference by the time interval (in years) between the two PSA tests.

The formula for calculating PSA velocity is as follows:

PSA Velocity (ng/mL/year) = (Later PSA - Initial PSA) / Time Interval
For example, if a man's initial PSA level was 2.5 ng/mL, and a subsequent test taken 12
months later showed a PSA level of 3.8 ng/mL, the PSA velocity would be:
PSA Velocity = (3.8 ng/mL - 2.5 ng/mL) / 1 year = 1.3 ng/mL/year
Interpretation of PSA Velocity:
A low or stable PSA velocity: If the rate of change in PSA levels is minimal or relatively
stable over time (usually less than 0.35 ng/mL/year), it is generally considered a
reassuring sign. It suggests that there is likely no rapid growth of prostate tissue or
cancerous cells.
A high PSA velocity: A significant increase in PSA levels over a short period (e.g., PSA
velocity greater than 0.75 ng/mL/year) may indicate a higher risk of prostate cancer.
However, a high PSA velocity alone is not enough to definitively diagnose prostate

36
cancer. Further evaluation, including a prostate biopsy, is required to confirm the
presence of cancer.
Limitations of PSA Velocity:
PSA velocity may be influenced by various factors, including benign prostatic conditions
(e.g., benign prostatic hyperplasia - BPH), urinary tract infections, prostate manipulation
(e.g., catheterization, biopsy), and certain medications. These factors can lead to
fluctuations in PSA levels and affect the accuracy of PSA velocity as a prostate cancer
marker.
The use of PSA velocity as a standalone screening tool for prostate cancer is not
recommended. It is best used in combination with other parameters, such as the total PSA
level, age-specific PSA reference ranges, digital rectal examination (DRE) findings,
family history, and risk calculators, to assess an individual's overall risk of prostate
cancer.
While PSA velocity can provide valuable information, it is not a substitute for prostate
biopsy when cancer is suspected. A biopsy remains the definitive method for diagnosing
prostate cancer.
The interpretation of PSA velocity data should be addressed with a licensed healthcare
practitioner, as with any medical test or screening tool. They can take into account
specific aspects and provide pertinent recommendations for further examination and
therapy.

37
CHAPTER THREE
3.0 CONCLUSION
In conclusion, the detection of PSA utilizing various manufactured electrodes represents a
dynamic and promising area in the field of biomedical research and diagnostics. To increase the
sensitivity, specificity, and effectiveness of PSA detection, we have looked at a variety of
electrode fabrication processes throughout this session, each with their own advantages and
difficulties.

Traditional bulk electrodes have evolved into nanomaterial-based electrodes, microfabricated

sensors, and even cutting-edge methods like biosensors and immunosensors. These
developments have cleared the way for PSA detection techniques that are more precise, quick,
and economical.

Furthermore, it is impossible to stress the importance of early PSA detection because it is crucial
to the early identification and treatment of prostate cancer, a disease that affects millions of
people globally. These electrode-based detection technologies have made it easier for medical
practitioners and researchers to make well-informed judgments and provide prompt therapies,
ultimately leading to better patient outcomes and survival rates.

Challenges persist, though, as with any scientific endeavor. Continued focus is needed in the
areas of standardizing fabrication procedures, improving sensitivity and specificity, and solving

38
problems with affordability and accessibility. In order to overcome these obstacles, collaboration
amongst interdisciplinary research teams made up of engineers, chemists, biologists, and doctors
will be essential.

In conclusion, PSA detection utilizing manufactured electrodes has advanced significantly and
has enormous potential for enhancing early identification and treatment of prostate cancer. We
may anticipate increasingly more advanced and efficient PSA detection techniques to emerge as
technology develops and interdisciplinary collaboration flourishes, bringing us closer to a future
in which prostate cancer is recognized and treated with more accuracy and success.

REFERENCES

 Xu, L., Wen, Y., Pandit, S., Mokkapati, V. R. S. S., Mijakovic, I., Li, Y., Ding, M., Ren,

S., Li, W., & Liu, G. (2019). Graphene-based biosensors for the detection of prostate

cancer protein biomarkers: a review. BMC Chemistry, 13(1).

https://doi.org/10.1186/s13065-019-0611-x

 Meryam Sardar, R. A. (2015). Enzyme Immobilization: An Overview on Nanoparticles

as Immobilization Matrix. Biochemistry & Analytical Biochemistry, 04(02).

https://doi.org/10.4172/2161-1009.1000178

 PUP ADDICT. (2019). PUP ADDICT. http://www.idealibrary.com

 Sueda, S. (2022). Antibody immobilization for immunosensing. Analytical Sciences,

38(1), 1–2. https://doi.org/10.1007/s44211-021-00019-w

 Zhao, Z., & Jiang, H. (2010, February 1). Enzyme-based Electrochemical Biosensors.

Www.intechopen.com; IntechOpen.

http://www.intechopen.com/books/biosensors/enzyme-based-electrochemical-biosensors

39
 Jolly, P., Formisano, N., & Estrela, P. (2015). DNA aptamer-based detection of prostate

cancer. Chemical Papers, 69(1). https://doi.org/10.1515/chempap-2015-0025

 Robert D. Crapnell, Nina C. Dempsey-Hibbert, Marloes Peeters, Ascanio Tridentec,

Craig E. Banks. Molecularly imprinted polymer based electrochemical biosensors:
Overcoming the challenges of detecting vital biomarkers and speeding up diagnosis.
www.elsevier.com/locate/talo
 Takita, S., Alexei Nabok, Lishchuk, A., Mussa, M. H., & Smith, D. P. (2023). Enhanced

Performance Electrochemical Biosensor for Detection of Prostate Cancer Biomarker

PCA3 Using Specific Aptamer. Eng, 4(1), 367–379. https://doi.org/10.3390/eng4010022

 Xu, L., Wen, Y., Pandit, S., Mokkapati, V. R. S. S., Mijakovic, I., Li, Y., Ding, M., Ren,

S., Li, W., & Liu, G. (2019). Graphene-based biosensors for the detection of prostate

cancer protein biomarkers: a review. BMC Chemistry, 13(1).

https://doi.org/10.1186/s13065-019-0611-x

 Mansuriya, B. D., & Altintas, Z. (2020). Applications of Graphene Quantum Dots in

Biomedical Sensors. Sensors, 20(4), 1072. https://doi.org/10.3390/s20041072

 Assari, P., Rafati, A. A., Feizollahi, A., & Joghani, R. A. (2020). Fabrication of a

sensitive label free electrochemical immunosensor for detection of prostate specific

antigen using functionalized multi-walled carbon nanotubes/polyaniline/AuNPs.

Materials Science and Engineering: C, 115, 111066.

https://doi.org/10.1016/j.msec.2020.111066

 Stenman, U. H., Leinonen, J., Zhang, W. M., & Finne, P. (1999). Prostate-specific

antigen. Seminars in Cancer Biology, 9(2), 83–93. https://doi.org/10.1006/scbi.1998.0086

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