sensors

Review
Multiplexed Prostate Cancer Companion Diagnostic Devices
Josephine Aidoo-Brown, Despina Moschou and Pedro Estrela *

Centre for Biosensors, Bioelectronics and Biodevices (C3Bio), Department of Electronic & Electrical Engineering,
University of Bath, Claverton Down, Bath BA2 7AY, UK; [email protected] (J.A.-B.); [email protected] (D.M.)
* Correspondence: [email protected]

Abstract: Prostate cancer (PCa) remains one of the most prominent forms of cancer for men. Since the
early 1990s, Prostate-Specific Antigen (PSA) has been a commonly recognized PCa-associated pro-
tein biomarker. However, PSA testing has been shown to lack in specificity and sensitivity when
needed to diagnose, monitor and/or treat PCa patients successfully. One enhancement could in-
clude the simultaneous detection of multiple PCa-associated protein biomarkers alongside PSA,
also known as multiplexing. If conventional methods such as the enzyme-linked immunosorbent
assay (ELISA) are used, multiplexed detection of such protein biomarkers can result in an increase in
the required sample volume, in the complexity of the analytical procedures, and in adding to the cost.
Using companion diagnostic devices such as biosensors, which can be portable and cost-effective
with multiplexing capacities, may address these limitations. This review explores recent research
for multiplexed PCa protein biomarker detection using optical and electrochemical biosensor plat-
forms. Some of the novel and potential serum-based PCa protein biomarkers will be discussed in
this review. In addition, this review discusses the importance of converting research protocols into
multiplex point-of-care testing (xPOCT) devices to be used in near-patient settings, providing a more



personalized approach to PCa patients’ diagnostic, surveillance and treatment management.



Citation: Aidoo-Brown, J.; Moschou, Keywords: prostate cancer; multiplex point-of-care testing (xPOCT); protein biomarkers; companion
D.; Estrela, P. Multiplexed Prostate diagnostic devices
Cancer Companion Diagnostic
Devices. Sensors 2021, 21, 5023.
https://doi.org/10.3390/s21155023
1. Introduction
Academic Editors: Eiichi Tamiya and
Prostate cancer (PCa) is one of the most prevalent cancer types for men worldwide [1].
Mun’delanji Vestergaard
So far, prostate-specific antigen (PSA) has been considered to be an important biomarker
for PCa diagnostic testing. In 1994, the use of a PSA screening test in combination with a
Received: 16 June 2021
Accepted: 21 July 2021
digital rectal examination (DRE) was approved by the U.S. Food and Drug Administration
Published: 24 July 2021
(FDA) [2]. The PSA screening test is a standard clinical diagnostic test, comprised of a blood
analysis for the quantification of PSA. According to established guidelines, serum PSA
Publisher’s Note: MDPI stays neutral
levels above 4 ng/mL provide an indication that PCa is present in an individual. However,
with regard to jurisdictional claims in
PSA levels, particularly those between 4 and 10 ng/mL, are referred to as the diagnostic
published maps and institutional affil- gray zone, in which elevated serum levels can be associated with other benign conditions,
iations. which can often be age-specific, such as benign prostatic hyperplasia (BPH) or prostatitis [3].
In addition to this, “normal” or “healthy” serum PSA levels (<4 ng/mL) can also be found
in PCa patients. Therefore, the PSA test is lacking in both sensitivity and specificity
for early detection of PCa. Leading to tentative misdiagnosis or needless and invasive
prostate biopsies or radical prostatectomies for numerous PCa patients [4,5]. Other PSA
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
derivatives have been calculated to improve PSA specificity, such as age-specific PSA
This article is an open access article
cut-offs, percentages between free PSA and total PSA (%fPSA), PSA density (PSAD) and
distributed under the terms and PSA velocity (PSAV) [5–10]. However, these attempts have not greatly increased the
conditions of the Creative Commons sensitivity and the specificity of the PCa diagnosis and treatment management. On the
Attribution (CC BY) license (https:// other hand, DRE testing normally has good specificity, e.g., DRE has been able to determine
creativecommons.org/licenses/by/ approximately 25% of clinically significant PCa patients who had been originally reported
4.0/).

Sensors 2021, 21, 5023. https://doi.org/10.3390/s21155023 https://www.mdpi.com/journal/sensors

Sensors 2021, 21, 5023 2 of 33

to display “normal” PSA serum levels [11,12]. However, this examination has a major
drawback of variability depending on the experience of the examiner [12–14].
Because of the variations in the sensitivity and specificity of PSA and/or DRE tests,
one significant research approach has been to simultaneously detect a panel of PCa protein
biomarkers, also known as multiplexing [15–21]. Biomarkers for PCa can be identified in a
variety of bodily samples, including prostatic tissue, serum and urine. Other biomarkers
that could be examined include DNA methylation, microRNAs, circulating tumor DNA
(ctDNA), metabolomics, volatile organic compounds (VOCs), and circulating tumor cells
(CTCs) [10,22–25]. However, because of the widespread clinical and commercial ease of
quantifying serum protein levels, especially when using traditional biomarker detection
methods, this review concentrates on serum PCa protein biomarkers [10]. Potential PCa-
associated protein biomarkers that have been identified (including those that have not
been validated to date) for diagnostic, prognosis, and predictive stages, will be discussed
in this review.
Conventional biomarker detection methods are used in clinical laboratory environ-
ments such as surface plasmon resonance, fluorescence analysis and enzyme-linked im-
munosorbent assay (ELISA) [26]. Of which ELISA is a typical gold-standard technique used
for single-analyte detection of protein biomarkers retrieved from patient’s samples such
as serum or urine [27]. However, conventional ELISA analysis is a lengthy and laborious
process, requiring highly qualified professionals [28]. Furthermore, it is not a suitable
method for a more reliable and tailored approach to PCa care and treatment management
when trying to achieve precise, accurate outcomes while using a minute sample volume to
detect several protein biomarkers [16,26,29].
Several biosensor platforms integrated with microfluidic systems have demonstrated
multiple benefits when compared to ELISA, so that results can be obtained easily, requiring
less steps and reducing costs [30]. This review aims to give an overview of the recent
advances in biosensor systems that simultaneously detect multiple PCa-related proteins
using optical or electrochemical detection techniques; it offers an insight into possible
and effective integrated systems that can be translated into multiplex point-of-care testing
(xPOCT) devices to be used in near-patient environments, such as hospitals, GP clinics or
within patient’s homes.

2. Potential PCa Protein Biomarkers

Although PSA (human kallikrein 3, hK3) is a serine protease produced by epithelial
cells inside the prostate gland, it is not specific to PCa [10,19,31,32]. Elevated levels of serum
PSA may be caused by other factors than adenocarcinoma of the prostate. In addition,
PCa can be present in men with low serum PSA levels (<4 ng/mL). However, as mentioned
previously, PSA derivatives have been found to be of some clinical use to increase the
sensitivity and specificity of PCa diagnosis [32,33].
Generally, multiple molecular isoforms of PSA circulate in serum [32,34]. For instance,
approximately 70% of PSA can be found in serum as complexed PSA (cPSA), in which
PSA is bound to serine protease inhibitors, such as α1-antichymotrypsin (ACT) and α2-
macroglobulin [35,36]. About 30% of PSA do not form complexes with serine protease
inhibitors, and are known as free PSA (fPSA), which in itself has several variants such as
proenzyme PSA (proPSA) and intact or inactive PSA (iPSA) [36,37]. fPSA has been shown
to be under-expressed in PCa patients relative to healthy patients or patients found to have
benign diseases. Low fPSA serum levels have also been correlated with aggressive PCa [38].
Combinations of both cPSA and fPSA, with other PSA molecular isoforms is known as total
PSA (tPSA) or simply PSA. tPSA and fPSA have been analyzed in combination, to calculate
the %fPSA of patients with tPSA serum levels within the diagnostic gray zone (between
4 and 10 ng/mL). It has been shown that a higher %fPSA suggests lower probability of
PCa on biopsy and raises the likelihood of an increase in PSA caused by BHP. However,
both the %fPSA and PSA screening tests are constrained in their analytical performance
to provide consistent diagnosis of PCa due to inter-assay variability [32,39]. In addition,
Sensors 2021, 21, 5023 3 of 33

more research into detecting multiple PSA isoforms using serum-based immunoassays has
led to the development and commercialization of the Prostate Health Index (PHI; Beckman
Coulter) and the 4-kallikrein score test (4Kscore® Test; OPKO Health), both of which are
used in clinical laboratories to help with the decision of whether an initial or repeat biopsy
should be performed [5].
The Prostate Health Index (PHI) was approved by the FDA in 2012 for PCa diag-
nosis and active surveillance of PCa patients. The PHI compares serum protein levels
of fPSA, tPSA, and a variant of pPSA called [−2]proPSA (or p2PSA) using the equation
(p2PSA/fPSA) × tPSA1/2 ) [5,10,37,40]. The PHI test is intended for males over the age
of 50 who have PSA values of less than 10 ng/mL, a normal DRE examination and are
planning or reconsidering a prostate biopsy [41]. The PHI has been proven to have a
diagnosis accuracy of 71% and a specificity of 26%, avoiding up to 40% of needless biopsies.
Despite the fact that it has been demonstrated to outperform just evaluating fPSA or tPSA
serum levels [5,10,37], the question of setting the cut off threshold for clinically significant
PCa patients remains unanswered [40]. Furthermore, the 4-kallikrein score test (4Kscore®
Test) is a prediction model that uses laboratory analysis along with clinical characteristics
such as age, prior prostate biopsy, and DRE results to provide the best prognosis for PCa
patients [5,10,37,40]. The laboratory test comprises the measurement of four kallikrein
proteins, including three PSA isoforms: fPSA, tPSA, and iPSA, as well as human kallikrein
2 (hK2), which is 80 percent homologous to PSA [10,37]. Overall, this test is for men who
have high PSA levels and a positive DRE examination result. It has been found in multiple
validation studies to eliminate needless biopsies while also being able to identify men
with clinically severe PCa. However, there are certain restrictions on when this test can be
performed. For example, it can only be conducted if men have not had a DRE in the last
96 h, or if they have not had any therapy or procedure for BHP symptoms [42]. Despite the
fact that these tests have been thoroughly confirmed, research suggests that one method to
improve their specificity is to select meaningful protein biomarkers other than PSA and
related isoforms [10].
Currently, many emerging and potential PCa protein biomarkers have been identified
(see Table 1), in particular relating to the diagnostic, prognostic or predictive stages of
a PCa patient [16]. As illustrated in Figure 1, the function of a biomarker is to act as a
biological indicator to determine any biological change that is contrary to normal biologi-
cal conditions [10,43,44]. The main feature that needs to be taken into consideration are
whether the biomarker has the ability to differentiate PCa from other benign prostatic
conditions (diagnostic biomarker). In addition, PCa biomarkers should be able to pro-
vide a forecast discriminating insignificant or indolent PCa from clinically significant or
aggressive PCa (prognostic biomarker). Furthermore, PCa protein biomarkers should be
able to give insight of the likely patient response through active surveillance or during
treatment (e.g., hormone therapy or chemotherapy), in order to proceed with the ideal
treatment pathway (predictive biomarker) [10]. Unfortunately, it is very unlikely that a
single biomarker will show all of these desirable characteristics, particularly because the
majority of the biomarkers referred to in Table 1 of this review and in other respective
reviews are not PCa-specific since they are associated with several other cancers or diseases.
At the same time, however, simultaneously detecting multiple biomarkers instead of using
single-analyte quantification methods may provide more accurate analysis within the
diagnostic, prognostic and predictive stages of PCa [30,43,45]. Further developments are
required to accurately determine the significance of each protein biomarker to be called a
PCa-associated biomarker in order to efficiently predict PCa-related results, tailored for
each PCa patient [43].
Sensors 2021, 21, 5023 4 of 33

Table 1. Selected candidate serum protein biomarkers with the potential of being detected within the diagnostic, prognostic and/or predictive stages of PCa.

Protein Type Biological Clinical Normal PCa Cut-Off PCa

Characteristics Relevance Serum Level Level Purpose Sample Ref
• Overexpression in prostate
• Involved in the body’s fatty cancer (PCa) tissues
Alpha- Methylacyl- Peroxisomal and • Potential for distinguishing Serum,
CoA Racemase mitochondrial acid synthesis and - 10.6 ng/mL Diagnostic/ Tissue and [3,43,46–51]
(AMACR) racemase benign prostatic hyperplasia Prognostic Urine
metabolism
(BPH) patients from
PCa patients
• Increased expression leads to
• Inflammation marker
Cluster of elevated concentration of
• Presented either as a monocyte in BPH tissues - Diagnostic Serum and
Differentiation Glycoprotein 1.9 µg/mL Tissue [52–55]
(CD)-14 membrane bound CD-14 or compared to PCa tissues
in its soluble form
• Linked to advanced PCa
• High CgA serum levels seen
in patients with PCa-resistant
• A part of the granin family
hormone therapy compared
of proteins to patients with BPH and
Chromogranin-A • Released by indolent PCa.
Pro-hormone - - Diagnostic/ Serum
peptide neuroendocrine (NE) cells • PCa patients with higher Prognostic [39,43,45–47,56]
(CgA)
in the prostate gland and CgA levels have lower
used as a cell marker for prognosis and survival
endocrine and NE cells
compared to those with
lower CgA levels
• Linked to early
Early Prostate Cell Nuclear matrix • Uncertain contribution Serum and
carcinogenesis and to predict - - Diagnostic [2,3,39,43,46,48]
Antigen (EPCA)-1 protein to nuclear morphology Tissue
repeated biopsy
• Connected to early
carcinogenesis
Nuclear matrix • Uncertain contribution Diagnostic/
EPCA-2 • Can differentiate between - - Serum [2,3,39,43,46,48]
protein to nuclear morphology Prognostic
indolent PCa and
aggressive PCa
• Aids for the transport of
• Up-regulation in patients
proteins through the
Type II Golgi with PCa Serum,
Golgi Membrane Golgi Apparatus. • PCa gene fusion protein Diagnostic/
membrane 54 ng/mL - Tissue, [48,52,57–59]
protein (GOLM)-1 • Also known as Golgi
• Suggested to be an
Prognostic and Urine
protein
phosphoprotein 2
aggressive PCa predictor
(GOLPH2) or (GP73)
Sensors 2021, 21, 5023 5 of 33

Table 1. Cont.

Protein Type Biological Clinical Normal PCa Cut-Off PCa

Characteristics Relevance Serum Level Level Purpose Sample Ref
• Secreted by epithelial cells
in the prostate gland • Highly expressed in prostate
• 80% homologous to PSA tissue, especially as PCa
but different in progresses to
Human Kallikrein Serine protease enzymatic activity advanced stages Diagnostic/ Serum and [31,35,39,43,45–
enzyme • Splits pro-PSA, • Studies have shown strong - -
2 (hK2) Prognostic Tissue 47,49,50,60]
producing PSA correlation with PCa-specific
• Also known as survival, but large cohort
Kallikrein-related peptide 2 validation studies required
(KLK2)
• Produced alongside • Elevated serum levels
IGFBP-3 • Associated with cancer
Insulin-like Growth hormone- • Involved in multiple development during
dependent cellular growth-related subclinical disease stages - Diagnostic Serum [39,43,45,47,49,52,
Growth Factor-1 160 ng/mL 55,61–64]
(IGF-1) polypeptides responses, including • Not a useful biomarker for
synthesis of DNA, RNA, early diagnosis or screening
and cellular proteins of PCa
• Under-expressed in
PCa patients
• Produced alongside IGF-1 • Suggested to limit the
• Binds to approximately 75 availability of IGF-1 and
IGF-Binding
Binding protein to 90% of circulating IGF-1, regulate apoptosis 3.7 µg/mL - Serum
Diagnostic/Prognostic [39,45,52,55,62–64]
Protein-3 (IGFBP-3)
in conjunction with the • Suggested to be a strong
acid-labile subunit predictor of significant PCa
compared to indolent PCa
• Elevated levels in PCa cells
• Associated with metastatic
and androgen
• Has variable effects on
independent PCa
Interleukin-6 (IL-6) immune and hematopoietic
and Receptor • Predictors of disease extent 0.006–0.02 0.02–1 Prognostic/
Serum
Cytokine mechanisms ng/mL ng/mL [2,31,43,47,48,65–69]
(IL-6R) • Produced at acute and in the progression and Predictive
chronic inflammation sites survival of PCa patients
• Soluble IL-6R has a stronger
correlation to disease
progression than IL-6
• Belongs to the chemokine • Lower serum levels in
(CXC) family, ligand 4 metastatic PCa patients, 5–10 µg/mL
Platelet Factor-4 compared with healthy or in serum 10–500
Chemokine (CXCL4) (2–10 ng/mL ng/mL Diagnostic Serum [52,68,70–74]
(PF-4)
• Released during indolent/clinically
in plasma)
stimulation of the platelet insignificant PCa patients
Sensors 2021, 21, 5023 6 of 33

Table 1. Cont.

Protein Type Biological Clinical Normal PCa Cut-Off PCa

Characteristics Relevance Serum Level Level Purpose Sample Ref
• Also known as Acpp or • Elevated in patients with PCa
prostatic specific acid metastasizing to the bone
Prostatic Acid Glycoprotein phosphatase (PSAP) • Potential biomarker for PCa >2.0 Diagnostic/ Serum and
- [32,45,48,75–77]
Phosphatase (PAP) enzyme • More research is needed to progressions and response of ng/mL Prognostic Urine
fully understand the advanced PCa to androgen
biological role deprivation therapy
• Also known as
Kallikrein-related
peptidase 3 (KLK3) or
human kallikrein 3 (hK3)
• Specifically produced by • Increased expression within
epithelial cells in the the prostate gland Diagnostic/
Prostate-Specific Serine protease Serum and [35,39,45,47,48,52,
enzyme prostate gland • Widely known PCa 1–4 ng/mL 4.0 ng/mL Prognostic/
Antigen (PSA) Urine 68,69,75,78]
• Has the biological role of biomarker; however lacking Predictive
seminal fluid liquefaction in sensitivity and specificity
• Androgen-regulated by
androgen response
elements
• Expressed in the epithelial
cells of the prostate
Type II integral • Also known as Folate
hydrolase 1, which has the
• Highly expressed in PCa cells 300–650
Prostate-Specific membrane ng/mL
glycoprotein function of folate hydrolase compared to BPH and
200–300 Diagnostic/ Serum and [2,43,46–
Membrane Antigen normal cells ng/mL [68,69] or Prognostic Urine 49,52,60,68,69,79]
(PSMA)
with cell surface • Involved in cell stress • Suggested role in 349.4–946.6
carboxypeptidase reaction, signal ng/mL [79]
PCa progression
function transduction,
cell migration,
and nutrient uptake
• Possible therapy target
• Specifically produced in • Increased expression
Prostate Stem Cell Membrane the prostate gland associated with higher Serum and
- - Prognostic [2,46,48,49]
Antigen (PSCA) glycoprotein • Involved in the regulation Gleason score, higher stage Tissue
of cell proliferation and in the presence of
aggressive PCa
Sensors 2021, 21, 5023 7 of 33

Table 1. Cont.

Protein Type Biological Clinical Normal PCa Cut-Off PCa

Characteristics Relevance Serum Level Level Purpose Sample Ref
• Involved in the
• Androgen receptor ligand
development and
associated with. The spread
preservation of prostate
of PCa
gland and seminal vesicles • Higher levels in aggressive Prognostic/
Testosterone Steroid hormone • Significant role in sexual than indolent cancer patients - - Serum [43,45,46,78]
Predictive
development and • More research needed to
anabolism fully understand its
• Acts on endocrine signal
clinical relevance
transduction
• Growth factor
• Suggested promotion of PCa
• Involved in cell cell progression
Transforming
proliferation, immune
• Associated with higher
- - Prognostic Serum
Growth Factor- ß1 Cytokine [2,31,43,47,49,50]
(TGF-ß1) response, differentiation, tumor grade, tumor invasion
and angiogenesis and metastasis in
PCa patients
• Plasminogen activator, • Highly expressed in PCa and
converting plasminogen BPH patients compared to
Urokinase
Plasminogen Serine protease into plasmin healthy patients
Activator (uPA) and • Plasmin activates protases • Correlated with aggressive - - Diagnostic/ Serum and [2,31,39,43,47,48,50,
and Receptor transmembrane PCa recurrences Prognostic Tissue 80,81]
receptors associated with the • Suggested as a predictor of
(uPAR) degradation of biochemical progression
extracellular matrix following surgery
• Vital endothelial cell
Vascular growth factor in controlling • Elevated concentration in 657 (±43)
Dimeric,
Endothelial heparin-binding the angiogenesis of the PCa patients pg/mL for - Prognostic Serum
Growth Factor VEGF-D [43,52,55,66,82,83]
protein tumor and • Suggested PCa angiogenesis
(VEGF) increase vascular ligand
permeability
Sensors 2021, 21, 5023 4 of 32
Sensors 2021, 21, 5023 8 of 33

Figure1.1.Desirable
Figure Desirablecharacteristics
characteristicsofofideal
idealPCa-associated
PCa-associated biomarkers.
biomarkers. Reproduced
Reproduced with
with permission
permission
fromref.
from ref.[43].
[43].Copyright
Copyright2010
2010Ivyspring
Ivyspring InternationalPublisher.
InternationalPublisher.

Table 1. Selected candidate3. Multiplexed

serum Detection with
protein biomarkers of PCa
theProtein Biomarkers
potential via Miniaturized
of being detected Biosensorprognos-
within the diagnostic, Systems
tic and/or predictive stages of PCa.
As previously stated, there has been a lot of focus on developing biosensors that detect
numerous PCa protein biomarkers
Biological Clinical
simultaneously, a process known as
Normal Se- PCa Cut-
multiplexing, in or-
PCa
Protein Type der to improve decision-making during therum critical (diagnostic,Sample Ref
Characteristics Relevance LevelPCaOffmilestones
Level Purpose prognostic,
and predictive stages).• Antibodies are the most extensively used bioreceptor for detecting
Overexpression
these protein biomarkers.inWhen prostateantibodies
cancer bind to their target antigen, whether via a
sandwich or competitive (PCa) tissues
assay, the overall set-up is referred to as an immunoassay
• Involved in the Serum, [84].
Alpha- Methylacyl- Peroxisomal •
Immunosensors involve
body’s fatty acid thePotential
use of for dis-
a biosensor platform in order to Diagnos-
monitor theTissue
immunoas-
[3,43,46–
CoA Racemase and mitochon- tinguishing be- - 10.6 ng/mL
say from which
synthesis binding events
and me- between
nign prostatic hy-
antibodies and their respective PCa
tic/Prognostic biomarker
and 51]
(AMACR) drial racemase
tabolism
can be evaluated using several transducers such as mass-sensitive, Urine
electrochemical or
perplasia (BPH)
optical transducers [84]. The focus
patients of this section is to highlight current research achieve-
from
PCa patients
ments, demonstrating the sensitive simultaneous detection of multiple serum PCa protein
biomarkers using optical • Increased
and expres-
electrochemical transducers. Several optical detection pro-
sion leads to ele-
•tocols have been devised to quantify certain target analytes, including luminescence [85],
Inflammation
vated concentra-
marker
surface-enhanced Raman scattering [86], and surface plasma resonance [87] detection
tion of monocyte Serum tech-
Cluster of Differen- •niques.
Presented either as research has been performed on the application of amperometric,
Additionally,
Glycoprotein in BPH tissues 1.9 µg/mL - Diagnostic and Tis- [52–55]
tiation (CD) -14 a membrane bound
voltammetric and impedimetric
CD-14 or in its sol-
comparedmethods
to PCa for multiplex electrochemical biosensorsue detection.
The advantages and tissues
disadvantages of using both optical and electrochemical techniques
uble form
are further assessed in• more Linked to ad-
detail by Roda et al. [88].
vanced PCa
Figure 2 describes •
various approaches to multiplexing, which include: the use of
High CgA serum
spatially separated detection sites
levels seenfor each biomarker; spatially divided regions within a
in pa-
channel network or electrode tientsarray;
with PCa-the use of several labels such as enzymes, metallic
•nanoparticles, magnetic resistant
A part of theorgranin microbeads;hor- and finally the use of spatially encoding on a single
family ofsurface
transducer mone therapy
proteins using multiple labels (also known as barcoding) [16,20,89]. Some of
• Released by neuro- compared to pa-
these multiplexing approaches have been shown to improve the sensitivity of the biosensor
endocrine (NE) tients with BPH
Chromogranin- A Pro-hormone during PCa protein detection and also have the potential to becomeDiagnos- commercially [39,43,45
available
cells in the prostate and indolent PCa. - - Serum
(CgA) peptide xPOCT devices for For example, some of the research –47,56]
tic/Prognostic
gland and used as PCa
a • treatment
PCa patients management.
with dis-
cussed
cellincludes
marker forthe
en- use of higher
low-cost
CgAtechnologies
levels to produce proof-of-concept prototypes
docrine
or the integration have lower
and NE of microfluidic prog- to provide automated operation of biosensor
systems
cells nosis and sur-
platforms while requiring lower sample volumes [90,91].
vival compared
to those with
lower CgA levels
• Linked to early
• Uncertain contribu- Serum
Early Prostate Cell Nuclear matrix carcinogenesis [2,3,39,43
tion to nuclear mor- - - Diagnostic and Tis-
Antigen (EPCA) -1 protein and to predict re- ,46,48]
phology sue
peated biopsy
 gle transducer surface using multiple labels (also known as barcoding) [16,20,89]. Some of
these multiplexing approaches have been shown to improve the sensitivity of the biosen-
sor during PCa protein detection and also have the potential to become commercially
available xPOCT devices for PCa treatment management. For example, some of the re-
Sensors 2021, 21, 5023
search discussed includes the use of low-cost technologies to produce proof-of-concept9 of 33
prototypes or the integration of microfluidic systems to provide automated operation of
biosensor platforms while requiring lower sample volumes [90,91].

Figure 2. 2.
Figure Schematic
Schematicto to
multiplexing approaches
multiplexing in in
approaches order to to
order simultaneously detect
simultaneously multiple
detect target
multiple target
analytes of interest.
analytes of interest.

3.1.3.1. Optical
Optical Detection
Detection Methods
Methods
3.1.1. Luminescence
3.1.1. Luminescence
Luminescence
Luminescence generates
generates a variety
a variety of of cold
cold light
light emissions,
emissions, asas it not
it is is not governed
governed bybythethe
rising temperatures, as seen with incandescent detection platforms [92]. Thus, this type of of
rising temperatures, as seen with incandescent detection platforms [92]. Thus, this type
optical
optical detection
detection technique
technique generally
generally involves
involves anan excited
excited molecule
molecule thatthat emits
emits light
light energy
energy
while returning to its electronic ground state [93]. In terms of multiplexed detection of of
while returning to its electronic ground state [93]. In terms of multiplexed detection
PCaPCa protein
protein biomarkers,
biomarkers, thethe fluorescent,
fluorescent, chemiluminescent
chemiluminescent andand electro-chemiluminescent
electro-chemiluminescent
methods will be discussed below.
methods will be discussed below.
Fluorescence
Fluorescence
Fluorescence is a type of photoluminescence that is initiated by the absorption of light
energy Fluorescence
(photons). This is physical
a type ofphenomenon
photoluminescence that is as
is also known initiated by the absorption
photoexcitation [93]. Pho- of
light energy (photons). This physical phenomenon is also known
todetectors are used to measure the changes in intensity after binding events of the targetas photoexcitation [93].
analyte and bioreceptor [94]. The labels only emit light at certain wavelengths when thethe
Photodetectors are used to measure the changes in intensity after binding events of
targetisanalyte
analyte found and[30].bioreceptor [94]. The labels only emit light at certain wavelengths when
theRong
analyte is found
et al. developed [30]. a fluorescent lateral flow immunoassay (LFIA) using dual-
Rong et al. developed
color magnetic-quantum dot nanobeads a fluorescent
(MQBs)lateral flow immunoassay
to detect fPSA and cPSA(LFIA) using dual-
simultaneously,
color magnetic-quantum dot nanobeads (MQBs) to detect fPSA and cPSA simultaneously,
as shown in Figure 3 [95]. Initially, protein biomarkers were attached to the capture anti-
as shown in Figure 3 [95]. Initially, protein biomarkers were attached to the capture
bodies modified with red (MQB625) and green (MQB525) colored MQBs, for fPSA and
antibodies modified with red (MQB625) and green (MQB525) colored MQBs, for fPSA
cPSA respectively, using an off-line capture protocol. After magnetic separation from un-
and cPSA respectively, using an off-line capture protocol. After magnetic separation from
bound MQBs, the respective capture antibodies (anti-fPSA and anti-cPSA), were used as
unbound MQBs, the respective capture antibodies (anti-fPSA and anti-cPSA), were used
fluorescent detection probes. Subsequently, the sample solution was introduced to the
as fluorescent detection probes. Subsequently, the sample solution was introduced to
the LFIA, and using capillary forces, the sample solution migrated to the sensor surface
immobilized with the monoclonal anti-tPSA detection antibodies to the nitrocellulose
membrane, forming a sandwich format. Fluorescent images were analyzed using a dual-
color strip readout integrated into a smartphone, as UV LED light stimulated the fluorescent
detection probes attached to the protein biomarkers. The limits of detection for fPSA and
cPSA in diluted fetal bovine serum were 0.009 and 0.087 ng/mL, respectively, within 1 h.
Further to this, the LFIA was able to distinguish between clinical samples obtained from
PCa and BPH patients when simultaneously detecting fPSA and cPSA in order to evaluate
the %fPSA. Moreover, the clinical sample results were well correlated with the reference
method, chemiluminescent microparticle immunoassay. It was concluded that the LFIA
 brane, forming a sandwich format. Fluorescent images were analyzed using a dual-color
strip readout integrated into a smartphone, as UV LED light stimulated the fluorescent
detection probes attached to the protein biomarkers. The limits of detection for fPSA and
cPSA in diluted fetal bovine serum were 0.009 and 0.087 ng/mL, respectively, within 1 h.
Sensors 2021, 21, 5023 Further to this, the LFIA was able to distinguish between clinical samples obtained10from
of 33
PCa and BPH patients when simultaneously detecting fPSA and cPSA in order to evaluate
the %fPSA. Moreover, the clinical sample results were well correlated with the reference
method, chemiluminescent microparticle immunoassay. It was concluded that the LFIA
prototype could
prototype could bebe specifically
specifically used
used as
as aa xPOCT
xPOCT device
device in
in low-resource
low-resource environments
environments
providing accurate diagnosis of PCa patients.
providing accurate diagnosis of PCa patients.

Figure 3.
Figure 3. Smartphone-based
Smartphone-based dual-color
dual-color fluorescent
fluorescent LFIA
LFIA reader;
reader; (A)
(A) Internal
Internal structure
structure of
of the
the smartphone
smartphone readout
readout device.
device.
MQB625 and MQB525 conjugates captured on the test line were excited by a 365 nm UV LED light source. Red and green
MQB625 and MQB525 conjugates captured on the test line were excited by a 365 nm UV LED light source. Red and
emission signals passed through a dual-band emission filter (524/628 nm) and an external plano-convex lens, before reaching
green emission signals passed through a dual-band emission filter (524/628 nm) and an external plano-convex lens, before
the smartphone CMOS sensor, (B) Depiction of the smartphone readout device. Reproduced with permission from ref. [95].
reaching
Copyright the smartphone
2019 Elsevier. CMOS sensor, (B) Depiction of the smartphone readout device. Reproduced with permission from
ref. [95]. Copyright 2019 Elsevier.
Chemiluminescence
Chemiluminescence
Chemiluminescence (CL) is initiated by a chemical reaction between at least two lu-
Chemiluminescence
minescent (CL) is initiated
reagents and is manipulated by fluid
by the a chemical reaction The
flow [92,94,96]. between
energy at produced
least two
luminescent reagents and is manipulated by the fluid flow [92,94,96]. The energy
by the reaction of chemical reagents together causes the production of light [97]. The most produced
by the reaction
common of chemical
example reagents together
of chemiluminescent causesinvolves
detection the production of lightinteraction
the chemical [97]. The most
be-
common example of chemiluminescent detection
tween luminol and horseradish peroxidase (HRP) [98]. involves the chemical interaction between
luminol
For and horseradish
instance, Tang etperoxidase (HRP) [98]. the use of this detection technique to
al. have demonstrated
detectFor
theinstance, Tang et PF-4
PCa biomarkers al. have
and demonstrated the use of 3D-printed
PSA using an automated this detection techniquear-
microfluidic to
detect the PCa biomarkers PF-4 and PSA using an automated 3D-printed
ray [99]. Using a touchscreen interface to operate the system’s pump, a sandwich format microfluidic
array [99]. Using a touchscreen interface to operate the system’s pump, a sandwich format
was constructed, by first immobilizing spotted arrays of poly L-lysine-coated glass slides
was constructed, by first immobilizing spotted arrays of poly L-lysine-coated glass slides
with capture antibodies that bind to its respective protein biomarkers. This was followed
with capture antibodies that bind to its respective protein biomarkers. This was followed
by detection antibodies which were attached to several horseradish peroxidase labels (pol-
by detection antibodies which were attached to several horseradish peroxidase labels
yHRP) forming Ab2-polyHRP conjugates. The CL reagents were introduced to the detec-
(polyHRP) forming Ab2 -polyHRP conjugates. The CL reagents were introduced to the
tion chamber after the flow of the wash buffer. From which luminol reacted with hydro-
detection chamber after the flow of the wash buffer. From which luminol reacted with
gen peroxide (H2O2) and was oxidized in the presence of HRP. The signal was measured
hydrogen peroxide (H2 O2 ) and was oxidized in the presence of HRP. The signal was
using a coupled charged device (CCD) camera. A detection limit of 0.5 pg/mL was
measured using a coupled charged device (CCD) camera. A detection limit of 0.5 pg/mL
achieved for PF-4 and PSA in diluted calf serum within 30 min. The accuracy of the results
was achieved for PF-4 and PSA in diluted calf serum within 30 min. The accuracy of the
was also confirmed by correlating results with ELISA assays using serum samples from
results was also confirmed by correlating results with ELISA assays using serum samples
non-PCa and PCa patients. Thus, it presents great opportunities to be used as a PCa
from non-PCa and PCa patients. Thus, it presents great opportunities to be used as a PCa
xPOCT diagnostic devicein
xPOCT diagnostic device inresource-limited
resource-limitedenvironments,
environments,because
becauseit it
is is not
not only
only re-usa-
re-usable
ble and fast but also cost-effective compared to the traditional
and fast but also cost-effective compared to the traditional ELISA. ELISA.
Jolly et al. demonstrated an aptamer-based ELISA that replaced capture antibodies
in a sandwich immunoassay with DNA aptamers for the quantification of fPSA and the
glycoprofiling of fPSA [100]. It has been suggested that glycoprofiling of fPSA could be used
to distinguish between indolent and aggressive forms of PCa. Thus, reducing unnecessary
biopsies and further treatments that may have a negative impact on PCa patients [101].
A detection antibody, HRP-labeled anti-fPSA, was used for fPSA quantification within a
single microchannel as shown in Figure 4. Whereas the parallel channel had the biotinylated
Sambucus nigra (SNA) lectin (a biological protein) with a complementary streptavidin-HRP
label, forming an aptamer-lectin assay for fPSA glycoprofiling. The optical changes that
 in a sandwich immunoassay with DNA aptamers for the quantification of fPSA and the
glycoprofiling of fPSA [100]. It has been suggested that glycoprofiling of fPSA could be
used to distinguish between indolent and aggressive forms of PCa. Thus, reducing unnec-
essary biopsies and further treatments that may have a negative impact on PCa patients
Sensors 2021, 21, 5023 [101]. A detection antibody, HRP-labeled anti-fPSA, was used for fPSA quantification 11 of 33
within a single microchannel as shown in Figure 4. Whereas the parallel channel had the
biotinylated Sambucus nigra (SNA) lectin (a biological protein) with a complementary
streptavidin-HRP label, forming an aptamer-lectin assay for fPSA glycoprofiling. The op-
occurred during the binding of the respective receptors to fPSA were measured using a
tical changes that occurred during the binding of the respective receptors to fPSA were
microfluidic CL sensor, via a microscopic CCD camera, after luminol had flowed into the
measured using a microfluidic CL sensor, via a microscopic CCD camera, after luminol
respective microchannels. Detection limits for fPSA and fPSA glycans were 0.5 and 3 ng/mL
had flowed into the respective microchannels. Detection limits for fPSA and fPSA glycans
in PBS, respectively. The detection limits are both relevant to the clinical ranges achieved
were 0.5 and 3 ng/mL in PBS, respectively. The detection limits are both relevant to the
using standard antibody-based immunoassays to evaluate the diagnosis or prognosis PCa
clinical ranges achieved using standard antibody-based immunoassays to evaluate the di-
patients [100].
agnosis or prognosis PCa patients [100].

Figure4.4.Illustration
Figure Illustrationofofmicrofluidic
microfluidicchannel
channelfabrication
fabricationscheme
schemefor
forthe
thequantification
quantificationand
and glyco-
glycopro-
profiling of fPSA. Reproduced with permission from ref. [100]. Copyright 2016 Elsevier.
filing of fPSA. Reproduced with permission from ref. [100]. Copyright 2016 Elsevier.

Zhaoetetal.
Zhao al.used
usedaadual-labeled
dual-labeled CL CL immunoassay
immunoassay to to simultaneously
simultaneouslymeasuremeasuretPSA tPSA
and fPSA from diluted human serum samples in just over 1 h
and fPSA from diluted human serum samples in just over 1 h [102]. A sandwich [102]. A sandwich immu-im-
noassay was
munoassay wasalso
alsoused,
used,ininwhich
whichcapture
capturemonoclonal
monoclonalantibodies
antibodieswerewerefirst
firstimmobilized
immobilized
ononthe
thesensing
sensingplatform.
platform. However,
However, two two different
different labels,
labels, HRP
HRP andand alkaline
alkalinephosphatase
phosphatase
(ALP), were used to differentiate between tPSA and fPSA detectionmonoclonal
(ALP), were used to differentiate between tPSA and fPSA detection monoclonalantibod-
antibod-
ies. The
ies. The HRP-labeled
HRP-labeled antibody
antibodyboundboundtotoboth cPSA
both cPSA andand
fPSA waswas
fPSA usedused
to determine the
to determine
amount of tPSA present in the sample. Whereas only fPSA was recognized
the amount of tPSA present in the sample. Whereas only fPSA was recognized by the by the ALP-
labeled antibody.
ALP-labeled As a result,
antibody. two chemiluminescence
As a result, two chemiluminescence reactions occurredoccurred
reactions during the de-
during
tection measurement as HRP reacted with luminol, ALP reacted with
the detection measurement as HRP reacted with luminol, ALP reacted with its respective its respective CL
substrate, 4-methoxy-4-(3-phosphate-phennyl)-spiro-(1,2-dioxetane-3,2′adamantane)
CL substrate, 4-methoxy-4-(3-phosphate-phennyl)-spiro-(1,2-dioxetane-3,2 adamantane) 0
(AMPPD).Detection
(AMPPD). Detectionlimits
limitsofof0.03
0.03and
and0.05
0.05ng/mL
ng/mLwerewerefound
foundfor fortPSA
tPSAandandfPSA.
fPSA.The
There-
results obtained from this assay were also correlated with commercial
sults obtained from this assay were also correlated with commercial chemiluminescent chemiluminescent
kitsusing
kits usingclinical
clinicalsamples.
samples. ItIt was
was concluded
concluded that
that this
this device
device would
wouldbe beuseful
usefulforforearly
early
diagnosis of PCa and could be used for routine clinical testing
diagnosis of PCa and could be used for routine clinical testing [102]. [102].
Electrochemiluminescence
In contrast to CL, electrochemiluminescence (ECL) is electrochemically generated, and
Electrochemiluminescence
therefore electron transfer at or near the working electrode is initiated and manipulated only
In contrast to CL, electrochemiluminescence (ECL) is electrochemically generated,
and therefore electron transfer at or near the working electrode is initiated and manipu-
lated only after the application of the potential [94,96,98,103]. From which the light intensity
emitted is detected due to the excited state of the reagents during the ECL reaction [92,96,104].
Sardesai et al. used the ECL to simultaneously detect PSA and IL-6 using a microwell
single-wall carbon nanotube (SWCNT) immunoarray [105]. The SWCNT forests were situ-
ated within the hydrophobic polymer walls formed on a pyrolytic graphite (PG) chip inked
with poly(butadine), in order to provide a conductive environment for ECL measurements.
The array also consisted of a sandwich format with capture antibodies and detection anti-
bodies. The detection antibodies were coated with tris(bipyridine)ruthenium(II) chloride
Sensors 2021, 21, 5023 12 of 33

([Ru(bpy)3 ]2+ ) doped with silica nanoparticles (Ab2 /RuBPY-SiNP). Both detection anti-
bodies for PSA and IL-6 were bound to the same RuBPY-SiNPs in this study. To measure
ECL, an electrolyte solution containing an ECL enhancer, tripropylamine (TrpA), initiated a
chemical reaction with [Ru(bpy)3 ]2+ at 0.95 V vs. Ag/AgCl. Once the potential was applied,
photoexcited [Ru(bpy)3 ]2+ was produced and was detected for 400 s using a CCD camera,
only when an intensity of light was emitted at 610 nm. Detection limits were 1 pg/mL
for PSA and 0.25 pg/mL for IL-6 in undiluted calf serum. Results using this array with
patients’ serum also correlated with the ELISA single-protein analyte kits. Following this
study, the same group adapted the microwell SWCNT immunoarray by integrating it with
a microfluidic system for the detection of the same protein biomarkers (PSA and IL-6),
which reduced the total assay time to just over an hour in comparison to three-hours when
using non-microfluidic arrays [106]. The microfluidic system consisted of three molded
polydimethylsiloxane (PDMS) channels which were situated on top of the chip and sup-
ported by a poly(methylmethacrylate) (PMMA) plate. The system also included a pump,
a sample injector and a switching value for directing solutions to their respective channels.
The authors achieved low detection limits for PSA (100 fg/mL) and IL-6 (10 fg/mL) in
calf serum. The microfluidic device required only 2.5 µL of serum samples to preform
triplicate analyses.
Kadimisetty et al. developed a 30-well microfluidic immunoarray using a low-cost
automated microprocessor to detect four PCa protein biomarkers in less than 40 min [107].
The microprocessor was integrated with printed circuit board (PCB)-controlled microp-
umps, which were connected to six PDMS channels, as shown in Figure 5. SWCNT forests
were also immobilized on the PG wafer to amplify the conductivity of the surface area.
In this research protocol, RuBPY-SiNPs were coated with two antibody mixtures to form
two duplex Ab2/RuBPY-SiNP detection labels, where label 1 was for PSA and IL-6 and
label 2 for PSMA and PF-4. Within 36 min, low detection limits of 50, 100, 10 and 10 fg/mL
Sensors 2021, 21, 5023 12 of 32
were achieved for PSA, PSMA, PF-4 and IL-6 in undiluted calf serum, respectively. Excellent
correlation was achieved with PCa patient serum compared to single-protein ELISA kits.

Figure
Figure5.5.AAmicrofluidic
microfluidicimmunoarray
immunoarray with a 30-well
with detection
a 30-well arrayarray
detection attached to PCB-controlled
attached mi-
to PCB-controlled
cropumps
micropumps andand
sample/reagent
sample/reagentcassette. The The
cassette. Arduino microcontroller
Arduino is the
microcontroller microprocessor
is the used
microprocessor to to
used
function the micropumps in order to perform the assay. Reproduced with permission from ref.
function the micropumps in order to perform the assay. Reproduced with permission from ref. [107]. [107].
Copyright 2015 American Chemical Society.
Copyright 2015 American Chemical Society.

Additionally, Kadimisetty et al. designed a 3D-printed supercapacitor-powered im-

munoarray to detect PSA, PSMA and PF-4 [108]. The supercapacitor was used to recharge
the sensor system using solar cells between ECL measurements. The simplicity of the
protocol reduced the cost of the immunoarrays’ materials, while achieving ultrasensitive
detection within 35 min, which is comparable to their previous work. Detection limits
of 300 fg/mL for PSA, 535 fg/mL for PSMA and 420 fg/mL for PF-4 were achieved in
serum. The device, as depicted in Figure 6, could be used as a xPOCT in a low-resourced
environment.
 Figure 5. A microfluidic immunoarray with a 30-well detection array attached to PCB-controlled mi-
Sensors 2021, 21, 5023 cropumps and sample/reagent cassette. The Arduino microcontroller is the microprocessor 13 of 33
used to
function the micropumps in order to perform the assay. Reproduced with permission from ref. [107].
Copyright 2015 American Chemical Society.

Figure 6.6. Illustration

Figure Illustration of
of 3D-printed
3D-printed supercapacitor-powered
supercapacitor-powered immunoarray
immunoarray using
using ECL
ECL detection
detection
technique. Reproduced with permission from ref. [108]. Copyright 2015 Elsevier.
technique. Reproduced with permission from ref. [108]. Copyright 2015 Elsevier.

Further to
Further to this,
this, Kadimisetty
Kadimisettyetetal. al.developed
developed another
anothercost-effective
cost-effective3D-printed
3D-printed immu-
im-
noarray to detect eight potential PCa protein biomarkers via ECL.
munoarray to detect eight potential PCa protein biomarkers via ECL. This included This included a 16 mi-a
crowell
16 detection
microwell chip, chip,
detection and aand microfluidic system
a microfluidic integrated
system with awith
integrated user-friendly touch
a user-friendly
screenscreen
touch interface, as depicted
interface, in Figure
as depicted 7. The 7.
in Figure touch
The screen interface
touch screen was used
interface wastoused
controlto
the automated
control micropump
the automated which iswhich
micropump connected to the microarray’s
is connected inlet port in
to the microarray’s to order
inlet port in to
deliver
to ordersamples
to deliverand reagents
samples andin areagents
timely manner [52].manner
in a timely The authors
[52]. used the method
The authors usedmen-the
tioned earlier
method mentionedin theearlier
Sardesai et al.
in the study et
Sardesai [106], which[106],
al. study involved
whichtheinvolved
use of four
the duplex
use of
Ab2/RuBPY-SiNP
four detection labels
duplex Ab2 /RuBPY-SiNP (label 1 for
detection PSA(label
labels and PSMA,
1 for PSAlabel
and2 for VEGF-D
PSMA, and
label PF-
2 for
4, label 3and
VEGF-D for CD-14 and IGF-1,
PF-4, label and label
3 for CD-14 and4IGF-1,
for GOLM-1 and4IGFBP-3).
and label for GOLM-1 Once theIGFBP-3).
and detection
antibodies
Once have been
the detection attachedhave
antibodies to their
beentarget protein,
attached TprA
to their solution
target was
protein, introduced
TprA solutiontowasthe
detection platform.
introduced The light
to the detection intensity
platform. Theoflight
the intensity
ECL reactions was reactions
of the ECL measuredwas using a CCD
measured
using
camera a CCD camera
located located
in a dark boxin asathedark box as the
potential waspotential
applied.was applied. In
In undiluted undiluted
calf calf
serum, ultra-
serum, ultra-low detection limits between 110 and 500 fg/mL were
low detection limits between 110 and 500 fg/mL were achieved for PSA, CD-14, GOLM-1, achieved for PSA,
CD-14, GOLM-1, IGFBP-3, IGF-1, PF-4, VEGF-D and PSMA within 25 min. The 3D-printed
immunoarray exhibited accurate recovery percentages of approximately 100 ± 14%, while
also achieving negligible antibody cross-reactivity between all eight proteins. The 3D-
printed immunoarray could distinguish between non-PCa and PCa patients. Additionally,
the authors suggested that this immunoarray could potentially be used to distinguish
between clinically insignificant and significant PCa patients. However, more tests using
human serum samples are needed to ensure that this is firmly concluded. Overall, it was
deduced that the easy-to-use immunoarray is cost-effective, with xPOCT characteristics,
especially when required in low-resourced environments [52].
 exhibited accurate recovery percentages of approximately 100 ± 14%, while also achieving
negligible antibody cross-reactivity between all eight proteins. The 3D-printed immuno-
array could distinguish between non-PCa and PCa patients. Additionally, the authors
suggested that this immunoarray could potentially be used to distinguish between clini-
cally insignificant and significant PCa patients. However, more tests using human serum
Sensors 2021, 21, 5023 samples are needed to ensure that this is firmly concluded. Overall, it was deduced that14 of 33
the easy-to-use immunoarray is cost-effective, with xPOCT characteristics, especially
when required in low-resourced environments [52].

Figure 7.
Figure 7. A
A3D-printed
3D-printedimmunoarray
immunoarray with touch
with screen
touch useruser
screen interface to control
interface ECL measurements.
to control ECL measurements.
A microfluidic array connected to a micropump is shown with dye-filled reagent chambers and
A microfluidic array connected to a micropump is shown with dye-filled reagent chambers and
graphite detection chip. Inset figures show multiple immunoassay steps along with messages to
graphite detection
inform the chip. Inset figures
user. Reproduced show multiple
with permission from immunoassay steps 2018
ref. [52]. Copyright alongAmerican
with messages to inform
Chemical
the user.
Society. Reproduced with permission from ref. [52]. Copyright 2018 American Chemical Society.

3.1.2.
3.1.2. Surface-Enhanced RamanScattering
Surface-Enhanced Raman Scattering
Surface-enhanced
Surface-enhanced Raman Ramanscattering
scattering (SERS)
(SERS) is another
is another optical
optical detection
detection technique
technique
used to demonstrate the analysis of multiple protein markers because
used to demonstrate the analysis of multiple protein markers because it is non-destruc- it is non-destructive,
photostable and sensitive
tive, photostable to the
and sensitive assessment
to the assessmentof of
specific biomarkers
specific biomarkers[109].[109].SERS
SERS hashas been
developed
been developedto increase
to increasethethe
intensity
intensityofofRaman scatteringby
Raman scattering bya factor
a factor of to
of up up10to 1012 [110].
12 [110].

The
The enhanced intensityisisenough
enhanced intensity enough toto measure
measure sufficiently
sufficiently the the changes
changes in plasmonic
in plasmonic reso- reso-
nance
nance ofof molecules adsorbedsingularly
molecules adsorbed singularlyon onorornear
nearroughened
roughened noble
noble metalmetal surfaces,
surfaces, such as
such
gold (Au)
as gold (Au)or or
silver
silver(Ag)
(Ag)[98,111–114]. Zhouetetal.al.developed
[98,111–114]. Zhou developed a multiplex
a multiplex SERS-based
SERS-based
immunoassay that
immunoassay thatdetected
detectedPSA,PSA,PSMAPSMA andandhK2
hK2[60]. A sandwich
[60]. A sandwich immunoassay
immunoassay was was
formed using
formed using silver
silvernanoparticles
nanoparticles (AgNPs)
(AgNPs) as as
thethe
platform for the
platform for immobilization
the immobilization of theof the
capture antibodies.
capture antibodies.The The SiC/Ag/Ag-NPs
SiC/Ag/Ag-NPs SERSSERS
substrate was attached
substrate to the detecting
was attached an-
to the detecting
antibodies to bind to the respective antigen. Using linear support vector machineal-(SVM)
tibodies to bind to the respective antigen. Using linear support vector machine (SVM)
gorithms, the
algorithms, thelimits
limitsofofdetections
detections forfor
PSA,
PSA,PSMA,
PSMA,andand hK2hK2
were 0.460.46
were fg/mL,
fg/mL,1.05 1.05
fg/mL fg/mL
and 0.67 fg/mL, respectively. Additionally, they achieved 70% accuracy
and 0.67 fg/mL, respectively. Additionally, they achieved 70% accuracy in distinguishing in distinguishing
between PCa
between PCa patients
patientswith withBHPBHPand andhealthy
healthypatients.
patients.TheThe
accuracy of the
accuracy ofdiagnosis of
the diagnosis of
healthy or BHP patients was 75% and 60%, respectively. In comparison, 50% accuracy was
healthy or BHP patients was 75% and 60%, respectively. In comparison, 50% accuracy
achieved to detect only the serum level of PSA. This SERS platform could be used for
was achieved to detect only the serum level of PSA. This SERS platform could be used for
diagnosing PCa patients in terms of providing clinical xPOCT.
diagnosing PCa patients in terms of providing clinical xPOCT.
Additionally, Chen et al. has developed a SERS-based vertical flow assay (VFA) for
the detection of PSA, carcinoembryonic antigen (CEA) and α-1-fetoprotein (AFP) [115].
CEA and AFP are cancer protein biomarkers that are not specific to PCa as they can be
found in a variety of cancer types [116]. Normally, conventional point-of-care VFAs are
paper-based gold-conjugated immunoassays and gold colloids. Instead, Raman dyes (RDs)
encoded core-shell SERS nanotags were used as detection probes to improve the precision
and sensitivity during detection measurements. The authors have achieved detection limits
of 0.37, 0.43, and 0.26 pg/mL for PSA, CEA and AFP respectively. It was concluded that
this platform could be used as a xPOCT device, in conjunction with a portable Raman
instrumentation, as a benchtop device in a hospital or GP clinic. This is because the SERS
immunoassay provides highly sensitive biomarker detection meanwhile the VFA platform
enables faster operation and simple analysis [115].
Sensors 2021, 21, 5023 15 of 33

In addition to this, Xiao et al. demonstrated the sensitivity and portability of a mul-
tiplex SERS-based immunoassay using an LFIA reader to also evaluate AFP, CEA and
PSA [114]. The LFIA reader was 3D printed and could be incorporated between a choice of
two multi-channel LFIA reaction columns, as shown in Figure 8. Type 1 of the LFIA column
(single-sample, multimarker reaction column) involved the use of a liquid drainage grove
which runs the same solution on all eight connected channels. Whereas the Type 2 LFIA
column (multisample, single-marker reaction column) consisted of sample holders for each
lateral strip, making it possible to detect a specific cancer biomarker on different strips
within the same column. In Figure 9, either Type 1 or Type 2 of the LFIA column was placed
on the holder inside the reader and operated via the stepper motor and two-axis translated
stage to rotate back and forth and move up and down at specific times. Therefore, it was
possible to perform SERS detection for each strip using the Raman probe to focus on the test
and control lines, as targeted immunocomplexes were formed. In addition, the photoelectric
switch corrected the position of each observation window to ensure that measurements
were carried out in an orderly manner. The SERS nanotags used were composed of gold
nanorods (AuNRs) and a Raman reporter molecule, 5,50 -dithiobis-(2-nitrobenzoic acid)
(DTNB), to match the laser wavelength of the excitation (785 nm). These AuNR−DTNB
plasmonic NPs were further functionalized with specific detection antibodies and BSA,
which provided biocompatibility and stability. The Type 1 column was used for evaluating
the LFIA reader’s specificity in which five antibodies were immobilized on the control line
of the strip. This involved the use of all three cancer biomarkers’ antibodies as well as the
antibodies specific to interfering inflammation biomarkers, C-reactive protein (CRP) and
Procalcitonin (PCT). The test line was found to visually darken only when the target cancer
biomarker was captured, regardless of the addition of interference protein biomarkers.
The visual interpretation of the test lines also corresponded to the SERS signal detected.
In addition, uniformity tests have shown that the device produces uniform, reliable and sta-
ble results. When using the Type 2 column, the overall process, which involved 20 repeats
for each of the eight strips used, took place within 18 min. Detection limits of 0.01 ng/mL
in PBS solution for all three cancer biomarkers were achieved, which was 1000 times more
sensitive than that acquired for visual signals. Lastly, clinical serum samples consisting of
either positive or negative samples were correctly detected for all three cancer biomarkers
using the Type 2 LFIA columns. The results of the clinical sample were also well cor-
related with ECL and demonstrated higher sensitivity compared to the ELISA analysis.
Overall, the SERS-based LFIA has the potential to be used as a xPOCT device in multiple
applications including the diagnosis and treatment management of PCa patients.
Sensors 2021, 21, 5023 An overview of recent developments of optical biosensors for multiplexed detection15 of 32
of PCa protein biomarkers is presented on Table 2.

Figure 8.
Figure 8. Detailed
Detailedfigure
figureofofthe
thetwo
two types of of
types multi-channel
multi-channel LFIA reaction
LFIA columns;
reaction (a) Single-sam-
columns; (a) Single-
ple, multimarker
sample, reaction
multimarker column
reaction (Type(Type
column 1); (b)1);
cross-sectional view ofview
(b) cross-sectional Typeof1 Type
LFIA 1column; (c) mul-
LFIA column;
tisample,
(c) single-marker
multisample, reaction
single-marker columncolumn
reaction (Type 2); (d) Type
(Type 2); (d)2 Type
column’s cross-sectional
2 column’s view. Repro-
cross-sectional view.
duced with permission
Reproduced from from
with permission ref. [114]. Copyright
ref. [114]. 2020 Elsevier.
Copyright 2020 Elsevier.
 Figure 8. Detailed figure of the two types of multi-channel LFIA reaction columns; (a) Single-sam-
Sensors 2021, 21, 5023 16 of 33
ple, multimarker reaction column (Type 1); (b) cross-sectional view of Type 1 LFIA column; (c) mul-
tisample, single-marker reaction column (Type 2); (d) Type 2 column’s cross-sectional view. Repro-
duced with permission from ref. [114]. Copyright 2020 Elsevier.

Figure Illustration
9. 9.
Figure Illustrationof of
portable SERS-based
portable SERS-based LFIA reader;
LFIA (a)(a)
reader; LFIA
LFIAstrip and
strip (b)(b)
and multi-channel LFIA
multi-channel LFIA
reaction column,
reaction column, (c)(c)
Detailed schematic
Detailed of of
schematic thethe
SERS-based
SERS-basedLFIALFIAreader
readerand (d)(d)
and thethe
completed
completedview
view
of of
thethe portable
portable reader.
reader. Reproduced
Reproduced with
with permission
permission from
from ref.ref. [114].
[114]. Copyright
Copyright 2020
2020 Elsevier.
Elsevier.

Table 2. Overview of recent developments of optical biosensors for multiplexed detection of PCa protein biomarkers with
indication of tests done with PCa patient samples.

Sensor Surface Linear Detection

Technique Biomarkers Modification Detection Label Range Limit of Detection Ref

Fluorescence fPSA Ab1 (monoclonal For fPSA: Ab2/MQB625 fPSA: 0.009 ng/mL
(PCa patient tPSA capture – [95]
samples) cPSA antibody) For cPSA: Ab2/MQB525 cPSA: 0.087 ng/mL
PSA: 0.5 pg/mL–
CL PSA
5 ng/mL * 0.5 pg mL−1 for
(PCa patient Ab1 Ab2 /poly HRP [99]
samples) PF-4: 0.5 pg/mL– both PSA and PF-4
PF-4
10 ng/mL *
For fPSA: Ab2 fPSA: 0.01 to
fPSA (Anti-fPSA)/HRPFor 50 ng/mL fPSA: 0.5 ng/mL
CL fPSA GOPTS/Apt fPSA glycans: [100]
glycans biotinylated fPSA glycans: 3 to fPSA glycans:
SNA/SA-HRP 50 ng/mL 3 ng/mL

CL For tPSA & fPSA: fPSA: 0.03 ng/mL

fPSA Ab1 (monoclonal Ab2/HRP
(PCa patient tPSA capture – [102]
tPSA
samples) antibody) For fPSA: Ab3/ALP tPSA: 0.05 ng/mL
Sensors 2021, 21, 5023 17 of 33

Table 2. Cont.

Sensor Surface Linear Detection

Technique Biomarkers Modification Detection Label Range Limit of Detection Ref

PSA: 1 pg/mL–
ECL PSA Ab2/RuBPY-SiNP PSA: 1 pg/mL
10 ng/mL *
(PCa patient SWCNT/Ab1 [105]
samples) (PSA and IL-6 conjugated IL-6: 0.1 pg/mL–
IL-6 IL-6: 0.25 pg/mL
to the same RuBPY-SiNPs) 2 ng/mL *
PSA: 100 fg/mL–
40 pg/mL
PSA Ab2/RuBPY-SiNP PSA: 100 fg/mL
ECL (100 fg/mL–
(PCa patient SWCNT/Ab1 10 ng/mL *) [106]
samples) IL-6: 0.5 fg/mL–
(PSA and IL-6 conjugated
IL-6 10 fg/mL IL-6: 10 fg/mL
to the same RuBPY-SiNPs)
(0.5 fg/mL–1 ng/mL *)
PSA: 100 fg/mL–
PSA PSA: 50 fg/mL
1 ng/mL *

PSMA PSMA: 100

ECL For Label 1 (PSA & IL-6) fg/mL–10 ng/mL * PSMA: 100 fg/mL
(PCa patient SWCNT/Ab1 and Label 2 (PSMA & [107]
samples) PF-4): Ab2 /RuBPY-SiNP PF-4: 100 fg/mL–
PF-4 PF-4: 10 fg/mL
5 ng/mL *
IL-6: 100 fg/mL–
IL-6 IL-6: 10 fg/mL
5 ng/mL *

ECL PSA For all proteins: PSA: 300 fg/mL

(PCa patient PSMA SWCNT/Ab1 Ab2 /RuBPY-SiNP 500 fg/mL– PSMA: 535 fg/mL [108]
samples) 10 ng/mL *
PF-4 PF-4: 420 fg/mL
PSA
PSMA
VEGF-D For Label 1 (PSA &
ECL PSMA), label 2 (VEGF-D For all proteins:
PF-4 & PF-4), label 3 (CD-14 & For all proteins:
(PCa patient SWCNT/Ab1 0.5 pg/mL– [52]
samples) CD-14 IGF-1), and label 4 110–500 fg/mL
(GOLM-1 & IGFBP-3): 10 ng/mL
IGF-1 Ab2 /RuBPY-SiNP
GOLM-1
IGFBP-3
PSA: 0.46 fg/mL–
PSA PSA: 0.46 fg/mL
478.93 ng/mL
SERS PSMA: 1.05 fg/mL–
(PCa patient SiC/Ag-
PSMA AgNPs/4-MBA/Ab2 PSMA: 1.05 fg/mL [60]
AgNPs/Ab1 113.4 ng/mL
samples)
hK2: 0.67 fg/mL–
hK2 hK2: 0.67 fg/mL
466.23 ng/mL

PSA For PSA: PSA:

AuNBA-Ag/Ab2 1 pg/mL–10 µg/mL PSA: 0.37 pg/mL
SERS For CEA: CEA:
(PCa patient CEA Ab1 Au4-MB-Ag/Ab2 10 pg/mL–1 µg/mL CEA: 0.43 pg/mL [115]
samples)
AFP For AFP: AFP:
Au4-NBT-Ag/Ab2 10 pg/mL–1 µg/mL AFP: 0.26 pg/mL

SERS PSA
(PCa patient - For all proteins:
CEA Ab1 AuNRs-DTNB/Ab2 /BSA [114]
samples) 10 pg/mL
AFP
Abbreviations: 4-MB = 4-Mercaptobenzonitrile, 4-MBA = 4-mercaptobenzoic acid, 4-NBT = 4-nitrobenzenethiol, Ab1 = capture antibody,
Ab2 = detection antibody, Ab3 = secondary detection antibody, AFP = α-1-fetoprotein, AgNP = silver nanoparticles, ALP = al-
kaline phosphatase, Apt = Deoxyribonucleic acid aptamer, BSA = Bovine Serum Albumin, CD-14 = Cluster of differentiation-14,
CEA = Carcinoembryonic antigen, CL = Chemiluminescence, cPSA = complexed PSA, DTNB = 5,50 -dithiobis-(2-nitrobenzoic acid),
ECL = Electrochemiluminescence, fPSA = free PSA, GOLM-1 = Golgi membrane protein-1, GOPTS = (3-Glycidyloxypropyl) trimethoxysi-
lane, hK2 = Human kallikrein 2, HRP = horseradish peroxidase, IGF-1 = Insulin-like Growth Factor-1, IGFBP-3 = Insulin-like Growth
Factor binding protein-3, IL-6 = Interleukin-6, MQB = magnetic-quantum dot nanobeads, NBA = Nile Blue A, PF-4 = Platelet Factor-4,
PSMA = Prostate-specific membrane antigen, RuBPY-SiNP = Tris(bipyridine)ruthenium(II) chloride, SA = Streptavidin, SERS = Surface-
enhanced Raman scattering, SiNP = silica nanoparticles, SNA = Sambucus nigra, SWNCT = single-wall carbon nanotube, tPSA = total PSA,
VEGF-D = Vascular endothelial growth factor. * Dynamic detection ranges.
Sensors 2021, 21, 5023 18 of 33

3.2. Electrochemical Detection Methods

3.2.1. Amperometric Techniques
Amperometry focuses on measuring the resulting current as a constant potential is
applied within the electrochemical cell [117,118]. Amperometric detection was used by
Chikkaveeraiah et al. to achieve multiplexed detection of PSA, PSMA, PF-4 and IL-6 [68].
Single-wall carbon nanotube forests (SWCNF) have modified the four working electrodes.
Each working electrode was then immobilized with one of the four respective capture
antibodies. After the capture antibodies bound to the protein biomarkers, they are attached
to detection antibodies to form a sandwich immunocomplex. PSA and PSMA detection
antibodies have been modified with HRP, whereas streptavidin-HRP (SA-HRP) labels were
used to modify the PF-4 and IL-6 biotinylated detection antibodies. Approximately 16
SA-HRP labels were attached to a single antibody in order to achieve a higher sensitivity
while detecting an electrochemical signal. This significantly amplified the amperometric
signal detected during binding events for PF-4 and IL-6, as their protein dynamic concen-
tration ranges are lower compared to PSA and PSMA. In the presence of the mediator,
hydroquinone, HRP catalyzed H2 O2 producing specific amperometric reduction peaks at
a voltage of −0.3 V, while detecting different concentrations of the respective biomarker.
This resulted in detection limits of 1, 10, 1 and 0.03 ng/mL for PSA, PSMA, PF-4 and IL-6
in diluted calf serum, respectively [68].
Moreover, using amperometric detection, Chikkaveeraiah et al. were able to achieve
lower detection limits of 0.23 pg/mL and 0.30 pg/mL for PSA and IL-6, respectively,
in diluted calf serum using a PDMS microfluidic-based platform [15]. Capture antibodies
were immobilized on eight working electrodes that had previously been deposited with
glutathione-decorated gold nanoparticles (GSH-AuNPs). In this study, superparamagnetic
nanoparticles conjugated with specific detection antibodies (~90,000 per nanoparticle) and
HRP labels (~20,000 labels per nanoparticle) were used for the off-line capture of the PCa
protein biomarkers in calf serum solution. Using a syringe, the modified superparamag-
netic nanoparticles solution flowed to the respective electrode surfaces. Following this,
hydroquinone and H2 O2 solutions were introduced to initiate an electrochemical reaction
that could be detected amperometrically. Additionally, the biosensor platform was well
correlated with ELISA in the use of patient serum samples, while also dramatically reduc-
ing manufacturing costs compared to conventional systems. A faster total analysis time
(1.15 h) was achieved, and a minute sample volume (5 µL) was required to obtain highly
sensitive and specific results.
Sharafeldin et al. used an offline capture method within a microfluidic electrochemical
immunoassay to simultaneously detect PSA and PSMA in undiluted calf serum [119]. In this
case, the working electrodes were modified with iron oxide nanoparticles (Fe3 O4 NPs) on
graphene oxide nanosheets, which were then decorated with specific capture antibodies
using 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC)/N- hydrox-
ysulfosuccinimide (NHS) chemistry. The microfluidic system was used to introduce serum
samples to initiate the respective binding events of the immobilized capture antibodies
to their specific protein biomarker. The bound protein biomarkers were magnetically
separated from the unbound biomarkers in the sample. From which the solution flowed
through the microfluidic system to the detection chamber, where the detection antibodies
attached to their respective protein biomarker. Fe3 O4 NPs performed similarly to HRP,
capable of catalyzing hydrogen peroxide to produce an amperometric signal. Detection
limits of 15 and 4.8 fg/mL were achieved for PSA and PSMA, respectively. The results
were comparable to previous studies using detection antibodies modified with magnetic
beads and HRP. Moreover, the immunoassay was well correlated with the ELISA method
when using patient serum samples.
Mercer et al. developed a microfluidic immunoarray platform, powered by a pro-
grammable Arduino microcontroller, capable of detecting eight PCa protein biomarkers
simultaneously, negating the need for a desktop or laptop [55]. Using the protocol de-
scribed by Otieno et al. [120], the carbon working electrodes were modified with a layer of
Sensors 2021, 21, 5023 19 of 33

poly(diallyldimethylammonium chloride) (PDDA), followed by GSH-AuNPs. The modi-

fied electrodes were then immobilized with sandwich immunocomplexes, consisting of
MP-Ab2-HRP conjugates as the detection antibodies. The immunoarray exhibited two
amperometry protein detection chambers, as depicted in Figure 10. The Arduino micro-
controller powered the automated processes of the microfluidic system, incorporating
several components, including valve actuators, a syringe pump, magnetic stirrers and
an electronic display. Amperometric detection limits of 140, 90, 15, 13, 130, 150, 90 and
15 pg/mL were achieved in serum for PSA, VEGF-D, erythroblast transformation specific
related gene (ERG), IGF-1, CD-14, IGFBP-3, pigment epithelium-derived factor (PEDF-1)
and GOLM-1, respectively, within 30 min [55]. ERG is over-expressed in patients with
PCa and contributes to PCa progression [2,121], whereas PEDF is suggested to exhibit
down-regulated serum levels in PCa patients, acting as an angiogenesis inhibitor. Overall,
the microfluidic immunoarray platform demonstrated the possibility of being used as a
Sensors 2021, 21, 5023
clinical xPOCT device, such as a hospital clinic, suitable for diagnosing and staging
19 of 32
PCa
progression [55].

Figure 10. Illustration of an automated microfluidic immunoarray platform, featuring; (a) Arduino
Figure 10. Illustration of an automated microfluidic immunoarray platform, featuring; (a) Arduino
Uno microcontroller, (b) syringe pump, (c) sample injector, (d) servo-actuated valves, (e) capture
Uno microcontroller,
chambers and magnetic (b)stirrers,
syringe(f)pump, (c) sample
detection injector,
chambers, and (g)(d)
LCDservo-actuated valves, (e)
displays. Reproduced capture
with
chambers
permissionand magnetic
from ref. [55]. stirrers,
Copyright (f)2019
detection chambers,
Wiley-VCH Verlag.and (g) LCD displays. Reproduced with
permission from ref. [55]. Copyright 2019 Wiley-VCH Verlag.
3.2.2. Voltammetric Techniques
3.2.2. Voltammetry
VoltammetricisTechniques
a sub-class of amperometry which measures the flow of electrons as
Voltammetry
a varied is swept
potential is a sub-class
acrossofthe
amperometry which[118,122].
working electrode measures theet
Tang flow of electrons
al. devised a as
acost-effective
varied potential is swept across
electrochemical the working
microfluidic electrode
immunoarray [118,122].
containing Tang
eight et al. devised a
miniaturized
ports in orderelectrochemical
cost-effective to achieve 256 individual working
microfluidic microelectrodes,
immunoarray simultaneously
containing detect-
eight miniaturized
ing PSA
ports alongside
in order PSMA,
to achieve 256IL-6 and PF-4,working
individual within one hour [123]. It simultaneously
microelectrodes, was noted that each
detecting
immunoarray contained 32 sensors, which were divided into four sections, for the respec-
tive protein biomarkers, as depicted in Figure 11. For simplicity during the DPV measure-
ments, each 32-sensor array had its own on-chip reference and counter electrodes. SAM
modified electrodes were attached to the hydrophobic wells to prevent cross-contamina-
tion of the antibodies on their respective surface during immobilization. Furthermore, the
Sensors 2021, 21, 5023 20 of 33

PSA alongside PSMA, IL-6 and PF-4, within one hour [123]. It was noted that each im-
munoarray contained 32 sensors, which were divided into four sections, for the respective
protein biomarkers, as depicted in Figure 11. For simplicity during the DPV measurements,
each 32-sensor array had its own on-chip reference and counter electrodes. SAM modified
electrodes were attached to the hydrophobic wells to prevent cross-contamination of the
antibodies on their respective surface during immobilization. Furthermore, the SAM layer,
composed of mercaptopropionic acid (MPA), was immobilized on the electrode surface,
followed by the attachment of capture antibodies using EDC/NHS chemistry. An off-line
capture protocol was established within a separate reservoir to attach different protein
concentrations to their respective biotinylated detection antibodies that were functional-
ized in conjunction to biotinylated HRP labels (~8500 HRP labels per nanoparticle onto
streptavidin coated magnetic nanoparticles). From which bound detection antibodies
Sensors 2021, 21, 5023 were introduced to the microelectrodes using the microfluidic system by means20ofofan 32 inlet

tube connected to the reagent reservoir. To load the reagents into the microfluidic system,
a syringe was connected to the outlet tubing of all eight of the immune arrays, effectively
microfluidic
detecting thesystem,
proteinabiomarkers.
syringe was connected
Hydroquinoneto the and
outletHtubing of all
2 O2 were eight of thetoim-
introduced the mi-
mune arrays, effectively detecting the protein biomarkers. Hydroquinone
crofluidic system for the measurement of differential pulse voltammetry and H 2O2 were
(DPV). For six
introduced
replicates of to the protein
eight microfluidic system for the
concentrations, the measurement of differential
limits of detections for PSA,pulse
PSMA, voltam-
PF-4 and
metry
IL-6 (DPV). For
in diluted calfsix replicates
serum were of
2, eight protein
0.15, 0.1 and concentrations, the limits ofThis
0.05 pg/mL, respectively. detections for
immunoarray
PSA, PSMA, PF-4 and IL-6 in diluted calf serum were 2, 0.15, 0.1 and 0.05
demonstrated high-throughput detection of multiple protein biomarkers at a low cost, pg/mL, respec-
tively. This immunoarray demonstrated high-throughput detection of multiple protein
using simple but highly sensitive equipment.
biomarkers at a low cost, using simple but highly sensitive equipment.

Figure11.
Figure 11. An
An electrochemical
electrochemical microfluidic
microfluidicimmunoarray:
immunoarray: (A) (A)
256 256
individual working
individual microelec-
working microelec-
trodes configuration; (B) 8 microfluidic immunoarrays are connected via miniaturized 8-port man-
trodes configuration; (B) 8 microfluidic immunoarrays are connected via miniaturized 8-port manifold;
ifold; (C) molded PDMS microfluidic channel, and (D) deconstructed view of the integrated micro-
(C) molded
fluidic PDMS microfluidic
immunoarray. channel,
Reproduced and (D) deconstructed
with permission from ref. [123].view of the 2016
Copyright integrated microfluidic
American Chem- im-
munoarray.
ical Society.Reproduced with permission from ref. [123]. Copyright 2016 American Chemical Society.

Pan
Panet
etal. also used
al. also usedDPV
DPVtotodetect
detectVEGF
VEGF and
and PSA PSA from
from serum
serum samples
samples of PCa
of PCa patients
patients
simultaneously
simultaneously [124].
[124]. AAthree-step
three-stepfabrication
fabrication process
process involving
involving metal-film
metal-film deposition,
deposition,
photolithography
photolithography and and metal
metaletching
etchingwas was used
used to to develop
develop thethe two-electrode
two-electrode system
system con- con-
sisting
sistingof
ofgold
gold working andcounter
working and counterelectrodes
electrodes
onon a glass
a glass slide.
slide. ForFor
the the completion
completion of theof the
three-electrode system, a separate silver/silver chloride (Ag/AgCl) reference electrode
such as. The graphene oxide modified working electrode was immobilized with VEGF-
specific DNA aptamers. The electrode surface was then introduced with the VEGF solu-
tion. Similarly, to the CL protocol of Jolly et al. [100], detection antibodies were then in-
Sensors 2021, 21, 5023 21 of 33

three-electrode system, a separate silver/silver chloride (Ag/AgCl) reference electrode

such as. The graphene oxide modified working electrode was immobilized with VEGF-
specific DNA aptamers. The electrode surface was then introduced with the VEGF solution.
Similarly, to the CL protocol of Jolly et al. [100], detection antibodies were then introduced
to the surface of the sensor. However, PSA and VEGF were analyzed on the same sensor
surface instead of using parallel microchannels. Detection antibodies for PSA and VEGF
(anti-PSA and anti-VEGF) were functionalized onto modified poly-L-lactide nanoparticles
(PLLA NPs) and then introduced to the electrode surface. In which the anti-VEGF antibod-
ies on the PPLA NPs bound to the VEGF protein immobilized on the electrode surface to
form a sandwich-based assay. After this, the biosensor was immersed in PSA solution that
also bound to the anti-PSA antibodies present on the PLLA NPs. This resulted in detection
limits of 50 pg/mL and 1 ng/mL for VEGF and PSA, respectively, and highly correlated
Sensors 2021, 21, 5023
with ELISA in the evaluation of samples from early staged PCa patients. 21 of 32
Alternatively, square wave voltammetry (SWV) was used by Akbari Jonous et al. to
simultaneously detect tPSA and fPSA, using a carbon working electrode with a sandwich-
based
resultedformat [125].
in detection Reduced
limits of 50 pg/mL graphene oxide
and 1 ng/mL forand
VEGF AuNPs
and PSA,were used toand
respectively, modify both the
highly correlated
capture with ELISA
and detection in the evaluation
monoclonal of samplesThis
antibodies. from early staged PCamagnified
significantly patients. the voltam-
metric Alternatively,
detection square
signalwave voltammetry (SWV)
as nanomaterials was used the
increased by Akbari
carbon Jonous et al. to conductivity,
electrode’s
simultaneously detect tPSA and fPSA, using a carbon working electrode with a sandwich-
resulting in faster electron transfer rates. Detection limits of 0.2 and 0.07 ng/mL were
based format [125]. Reduced graphene oxide and AuNPs were used to modify both the
determined for tPSAmonoclonal
capture and detection and fPSA, respectively.
antibodies. The biosensor
This significantly was the
magnified well correlated with the
voltam-
standard CL test
metric detection using
signal patients’ serum
as nanomaterials samples.
increased Theelectrode’s
the carbon authors suggested
conductivity,that it could be
resulting
used as ainPCafaster electron transfer
diagnostic POCT rates. Detection
device. limits of 0.2 and
Additionally, Liu0.07 ng/mL
et al. have were de-
developed a flexible
termined for tPSA and fPSA, respectively. The biosensor
PDMS 8 × 8 electrode immunoarray for multiplex electrochemical detection was well correlated with the of PSA, PSMA
standard CL test using patients’ serum samples. The authors suggested that it could be
and IL-6, as shown in Figure 12 [69]. The Au electrodes were used to form a sandwich-
used as a PCa diagnostic POCT device. Additionally, Liu et al. have developed a flexible
based
PDMS immunosensor, initiallyfor
8 × 8 electrode immunoarray immobilized with capture
multiplex electrochemical antibodies
detection functionalized with
of PSA, PSMA
magnetic
and IL-6, as beads.
shownIn in addition,
Figure 12 [69].detection
The Au antibodies
electrodes werewereusedfunctionalized
to form a sandwich-with AuNRs deco-
based with
rated immunosensor,
HRP (HRP-Ab initially 2immobilized
-AuNRs). with Cycliccapture antibodies functionalized
voltammetry (CV) was used withto measure the
magnetic beads.
resulting currentIn addition,
with low detection antibodies
detection limitswereoffunctionalized
0.1, 0.8 andwith 0.005AuNRs
ng/mLdeco-determined for
rated with HRP (HRP-Ab2-AuNRs). Cyclic voltammetry (CV) was used to measure the
PSA, PSMA and IL-6, respectively. The authors concluded that the microchip could be
resulting current with low detection limits of 0.1, 0.8 and 0.005 ng/mL determined for PSA,
used as
PSMA and a xPOCT device, exhibiting
IL-6, respectively. strengthsthat
The authors concluded duethetomicrochip
its versatility,
could beinused
terms as of fabrication,
modification
a xPOCT device, processes,
exhibiting and storage.
strengths due toItits
is versatility,
also less likely
in termstoofbe damaged
fabrication, compared to rigid
modi-
fication
glass processes, used
substrates and storage.
for the It is also less likely
fabrication of to be damaged
biosensor compared to rigid glass
platforms.
substrates used for the fabrication of biosensor platforms.

Figure 12. A flexible PDMS 8 × 8 electrode immunoarray; (A) Schematic of preparing the microchip
Figure A flexible
12. the
along with PDMS
sensor surface 8 × 8 electrode
modifications; immunoarray;
(B) Illustration (A) Schematic
of the detection of preparing
of PSA, PSMA and IL- the microchip
6, withwith
along control
themeasurements, in one
sensor surface microchannel. Reproduced
modifications; with permission
(B) Illustration from ref. [69].
of the detection of PSA, PSMA and
Copyright 2014 American Chemical Society.
IL-6, with control measurements, in one microchannel. Reproduced with permission from ref. [69].
Copyright 2014 American
3.2.3. Impedimetric Chemical Society.
Techniques
Impedance-based biosensors are label-free and highly sensitive electrochemical de-
tection techniques that reduce the number of reagents required and hence reduce the over-
all analysis time [4,94]. Electrical impedance spectroscopy evaluates the capacitive or re-
sistive behavior established from the charges separated at the electrode-electrolyte inter-
face [94,117]. Impedance-based biosensors require the application of a low sinusoidal ac
Sensors 2021, 21, 5023 22 of 33

3.2.3. Impedimetric Techniques

Impedance-based biosensors are label-free and highly sensitive electrochemical de-
tection techniques that reduce the number of reagents required and hence reduce the
overall analysis time [4,94]. Electrical impedance spectroscopy evaluates the capacitive
Sensors 2021, 21, 5023 or resistive behavior established from the charges separated at the electrode-electrolyte 22 of 32
interface [94,117]. Impedance-based biosensors require the application of a low sinusoidal
ac voltage (typically 5–10 mV) at a specific frequency [94,118]. The voltage perturbation is
used
used to to demonstrate
demonstratethe thebiological
biologicalbinding
bindingevents
eventsthat
thatoccur
occurat atthe
thesurface
surfaceofofthe
theelectrode
electrode
by
by evaluating
evaluating the
the charge
charge flow
flow [117].
[117]. The
The charge
charge flow
flowororelectrical
electricalsignal
signalcan
canbebemodeled
modeled
using
using thethe Randles
Randles equivalent
equivalent circuit,
circuit,asasdepicted
depictedin inFigure
Figure1313[126].
[126].The
Thecharge
chargeresistance
resistance
at
at the
the interfacial
interfacial layer
layer of
of the
the working
working electrode
electrode isis known
known as as the
the charge
charge transfer
transfer resistance,
resistance,
RRCT . Within the circuit, the
CT. Within the circuit, the RCTCT R is parallel to the capacitance, C
is parallel to the capacitance, CDL, which DL , which describes the
describes the elec-
electrode-electrolyte interface’s electrical double layer. Additionally, in series
trode-electrolyte interface’s electrical double layer. Additionally, in series to the RCT,CT to the R W,
W represents the Warburg diffusion coefficient
represents the Warburg diffusion coefficient and RSOLSOL and R is to demonstrate the
is to demonstrate the uncompen- uncom-
pensated resistance
sated resistance of of
thethe solution[127].
solution [127].Impedance
Impedancedatadataare
are generally
generally represented
represented using
using
Nyquist
Nyquist or or Bode
Bode plots
plots [128].
[128].

Figure 13. Randles equivalent circuit to

Figure 13. to the
the model
model the
the charge
charge flow
flowduring
duringelectrochemical
electrochemicalimpedance
impedance
spectroscopy
spectroscopy detection,
detection, RRCT representsthe
CTrepresents thecharge
charge transfer
transfer resistance,
resistance, RSOL
RSOL denotes
denotes the uncompen-
the uncompensated
solution
sated resistance,
solution CDL represents
resistance, the capacitance
CDL represents and W signifies
the capacitance and Wthe Warburg
signifies thediffusion
Warburgcoefficient.
diffusion
coefficient.
Chiriacò et al. designed an impedimetric dual-labeled microfluidic biosensor based
Chiriacò
on the et al. designed
conventional an impedimetric
ELISA method [129]. The dual-labeled
PDMS-basedmicrofluidic
microfluidicbiosensor
system had based
two
on the conventional
chambers, as shownELISA method
in Figure [129].chamber
14. Each The PDMS-based
consisted microfluidic
of modified system had two
SAM electrodes
chambers, as shown
with anti-fPSA in Figure antibodies,
and anti-tPSA 14. Each chamber consisted
respectively, usingofEDC/NHS
modified SAM electrodes
chemistry. Anti-
with anti-fPSA and anti-tPSA antibodies, respectively, using
tPSA capture antibodies could be attached to both biomarkers, fPSA and cPSA, bothEDC/NHS chemistry. Anti-of
tPSA
whichcapture
have the antibodies
same epitopecould be attached
recognition onto both
their biomarkers,
surfaces. fPSA andaccurate
This provided cPSA, both
%fPSA of
which have the same
measurements, epitope
therefore, recognition
in order on their
to diagnose surfaces.
and also toThis provided
distinguish PCaaccurate %fPSA
patients from
measurements,
other conditions. therefore,
The fPSA in and
order to diagnose
cPSA solutionsand also to
flowed intodistinguish PCa patients
their respective chambers.from A
other conditions. The fPSA and cPSA solutions flowed into their respective
solution containing the electrochemical redox probe, [Fe(CN)6] , was then used during
3–/4– chambers. A so-
lution containingimpedance
electrochemical the electrochemical
spectroscopy redox probe,
(EIS) [Fe(CN)6 ]3–/4–
measurements. , was
It was thenthat
found used during
following
electrochemical impedance spectroscopy (EIS) measurements. It
the functionalizing of the electrode with the SAM layer and subsequent binding of thewas found that following
the functionalizing
antibodies of the electrode
to their respective with the
biomarker, theSAM layer increased.
RCT value and subsequent bindingtransfer
The electron of the
antibodies to their respective biomarker,
from the bulk solution to the working electrodes the R value increased. The electron
CTwas therefore restricted. Sensitive detec- transfer
from the bulk solution to the working electrodes
tion limits of approximately 1 ng/mL in PBS solution was therefore
were achievedrestricted. Sensitive
for both detec-
biomarkers.
tion limits of approximately 1 ng/mL in PBS solution were achieved
Additionally, the authors were able to simultaneously detect fPSA and cPSA in order to for both biomarkers.
Additionally,
evaluate %fPSA. the If
authors wereisable
the %fPSA lowerto simultaneously
than cut-off level detect fPSA
(<25%), and
this cPSA in order
determined to
that the
evaluate %fPSA. If the %fPSA is lower than cut-off level (<25%), this determined that the
patient has PCa. Using two solutions with fixed %fPSA (50 and 20%), the biosensor was
patient has PCa. Using two solutions with fixed %fPSA (50 and 20%), the biosensor was
able to distinguish between the two solutions, calculating the %fPSA to be 42% and 19%,
able to distinguish between the two solutions, calculating the %fPSA to be 42% and 19%,
respectively. This platform therefore has the potential to distinguish BHP patients from
respectively. This platform therefore has the potential to distinguish BHP patients from
PCa patients. Thus, using this simple fabrication process, the overall analysis time has not
PCa patients. Thus, using this simple fabrication process, the overall analysis time has not
only been shortened compared to the conventional ELISA method, but is also cost-effec-
only been shortened compared to the conventional ELISA method, but is also cost-effective
tive using a simple manufacturing process.
using a simple manufacturing process.
Additionally, Pihíková et al. reported a label-free impedimetric biosensor capable of
detecting PSA and PSA glycans at the same time [130]. As mentioned previously, research
has been conducted on how changes in the conformation of PSA glycosylation can be
linked to PCa progression. Primarily, the capture antibodies were immobilized to the
SAM layer, composed of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol, on the
Sensors 2021, 21, 5023 23 of 33

Additionally, Pihíková et al. reported a label-free impedimetric biosensor capable of

detecting PSA and PSA glycans at the same time [130]. As mentioned previously, research
has been conducted on how changes in the conformation of PSA glycosylation can be
linked to PCa progression. Primarily, the capture antibodies were immobilized to the
SAM layer, composed of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol, on the
Sensors 2021, 21, 5023
surface of the sensor, using EDC/NHS chemistry. This allowed the PSA to attach to23the of 32
capture antibodies. Mass spectrometry is typically used for the analysis of PSA glycans.
However, the lectin, SNA, was used by the impedimetric biosensor to detect glycosylated
PSA forms. The lectins specifically attached to the terminal sialic acid present on PSA,
toPSA
formforms. The lectins
a sandwich specifically
format. [Fe(CN)attached
6]
to the
3–/4– was usedterminal sialic acid present
as an electroactive redox on PSA,
probe forto
form a sandwich format. [Fe(CN) 6]3–/4– was used as an electroactive redox probe for EIS
EIS measurements. Sensitive detection limits of 4 aM (about 0.13 fg/mL) were achieved for
measurements.
both PSA and PSA Sensitive
glycans.detection limits of 4 aM (about 0.13 fg/mL) were achieved for
both PSA and PSA glycans.

Figure14.
Figure 14.(a)
(a)Schematic
Schematicrepresentation
representationofofPSA
PSAantigens-related
antigens-related(cPSA
(cPSAand
andfPSA)
fPSA)and
and(b)
(b)device
device
composed of two chambers for detecting the antigens: (c) one chamber is functionalized with anti-
composed of two chambers for detecting the antigens: (c) one chamber is functionalized with anti-
fPSA antibodies (Chamber 1) and the other one with anti-tPSA antibodies (Chamber 2). Reproduced
fPSA antibodies (Chamber 1) and the other one with anti-tPSA antibodies (Chamber 2). Reproduced
with permission from ref. [129]. Copyright 2013 Royal Society of Chemistry.
with permission from ref. [129]. Copyright 2013 Royal Society of Chemistry.
Diaz Fernandez et al. developed a dual aptamer-based impedimetric biosensor, com-
Diaz Fernandez et al. developed a dual aptamer-based impedimetric biosensor,
prised of two adjacent nanostructured gold electrodes that detected both PSA and glyco-
comprised of two adjacent nanostructured gold electrodes that detected both PSA and
sylated PSA (PSAG-1) using anti-PSA and PSAG-1 aptamers [131]. A SAM layer of 11-
glycosylated PSA (PSAG-1) using anti-PSA and PSAG-1 aptamers [131]. A SAM layer
amino-1-undecanothiol was applied to the gold working electrodes. Mercaptohexanol
of 11-amino-1-undecanothiol was applied to the gold working electrodes. Mercaptohex-
(MCH)
anol was was
(MCH) usedused
as a as
blocking
a blocking agentagent
or backfiller before
or backfiller AuNPs
before were were
AuNPs introduced to im-
introduced
mobilize the SAM layer. After that, another SAM layer was applied
to immobilize the SAM layer. After that, another SAM layer was applied to the surface, to the surface, con-
sisting of a 1:100 ratio of the relevant aptamer (anti-PSA/PSAG-1)
consisting of a 1:100 ratio of the relevant aptamer (anti-PSA/PSAG-1) and MCH. In di- and MCH. In diluted
serum,
luted the aptasensor
serum, the aptasensorhad detection limits limits
had detection of 0.64ofand 0.26
0.64 andng/mL for PSAfor
0.26 ng/mL andPSAPSAG-1,
and
respectively. When impedimetric detection with the PSAG-1
PSAG-1, respectively. When impedimetric detection with the PSAG-1 aptamer was used aptamer was used to detect
torecombinant PSA (rPSA),
detect recombinant the signal
PSA (rPSA), rise was
the signal rise lower than than
was lower when detecting
when detectinghuman
human PSA
(hPSA). When using the anti-PSA aptamer to detect both rPSA
PSA (hPSA). When using the anti-PSA aptamer to detect both rPSA and hPSA, a similar and hPSA, a similar signal
increase
signal (96%) (96%)
increase was seenwasbetween the twothe
seen between PSA twoproteins. As a result,
PSA proteins. As aPSAG-1
result, has
PSAG-1been has
con-
firmed
been as the aptamer
confirmed that canthat
as the aptamer recognize PSA’s glycosylated
can recognize sites. Human
PSA’s glycosylated serum albumin
sites. Human serum
was shown to have a very little interference, indicating that this platform
albumin was shown to have a very little interference, indicating that this platform is overall is overall selective
to PSA. to
selective Furthermore, the glycan
PSA. Furthermore, thescore
glycan (GS) was(GS)
score calculated using clinical
was calculated using serum
clinicalsamples.
serum
samples. The GS is the ratio between the concentration of the glycosylated PSAwith
The GS is the ratio between the concentration of the glycosylated PSA (detected PSAG-
(detected
with PSAG-1 aptamer) to tPSA (detected with anti-PSA aptamer), multiped by 100. In com-to
1 aptamer) to tPSA (detected with anti-PSA aptamer), multiped by 100. In comparison
benign to
parison and healthy
benign andpatients
healthy(values
patientsbetween
(values22 and 37),22a and
between clear37),
correlation was discovered
a clear correlation was
between the
discovered GS andthe
between theGSknown diagnosis
and the knownofdiagnosis
PCa patients (values
of PCa between
patients 82 and
(values 86), as
between
82illustrated
and 86), inas Figure 15, rather
illustrated in Figure than15,
looking
rather at thelooking
than concentrations of the analytesofinde-
at the concentrations the
pendently.
analytes The authors The
independently. came to the came
authors conclusion
to the that this platform
conclusion that thismight be used
platform mightto be
im-
prove
used to patient
improvePCa diagnosis
patient while also
PCa diagnosis minimizing
while the number
also minimizing of unnecessary
the number biopsies
of unnecessary
performed.
biopsies performed.
An overview of recent developments of electrochemical biosensors for multiplexed
detection of PCa protein biomarkers is presented on Table 3.
Sensors 2021, 21, 5023 24 of 33

Sensors 2021, 21, 5023 24 of 32

An overview of recent developments of electrochemical biosensors for multiplexed
detection of PCa protein biomarkers is presented on Table 3.

Figure
Figure 15.
15. Illustration
Illustration of
of anti-PSA
anti-PSA and
and PSAG-1
PSAG-1 aptamers
aptamers used
used in
in the
the dual
dual aptamer-based
aptamer-based impedimetric
impedimetric biosensor
biosensor to
to detect
detect
PSA
PSA and PSA glycans. Using clinical serum samples, the EIS measurement were used to measure the glycan score (GS),
and PSA glycans. Using clinical serum samples, the EIS measurement were used to measure the glycan score (GS),
which is the ratio between the concentration of the glycosylated PSA (detected with PSAG-1 aptamer) to tPSA (detected
which is the ratio between the concentration of the glycosylated PSA (detected with PSAG-1 aptamer) to tPSA (detected
with anti-PSA aptamer), multiped by 100. According to the graphical data, the GS can be used to distinguish known PCa
with anti-PSA aptamer), multiped by 100. According to the graphical data, the GS can be used to distinguish known PCa
patients from benign and healthy patients. Reproduced with permission from ref. [131]. Copyright 2020 Elsevier.
patients from benign and healthy patients. Reproduced with permission from ref. [131]. Copyright 2020 Elsevier.
Table 3. Overview of recent developments of electrochemical biosensors for multiplexed detection of PCa protein bi-
Table 3. Overview
omarkers of recentofdevelopments
with indication of electrochemical
tests done with biosensors for multiplexed detection of PCa protein biomarkers
PCa patient samples.
with indication of tests done with PCa patient samples.
Linear Detection
Technique Biomarkers Sensor surface modification Detection Label Limit of Detection Ref
Sensor surface Range
Linear Detection
Technique Biomarkers Detection Label Limit of Detection Ref
PSA modification PSA: Range
1–40 ng/mL PSA: 1 ng/mL
PSMA PSA PSMA: 10–250
PSA: 1–40 ng/mL
ng/mL PSMA:
PSA: 110ng/mL
ng/mL
Amp For PSA & PSMA:
PF-4 PF-4: 1–40 ng/mL
PSMA: 10–250 PF-4: 1 ng/mL
(PCa pa- PSMA SWCNF/Ab1 For PSA & PSMA: Ab2/HRP PSMA: 10 ng/mL
For IL-6: 50–500
ng/mL pg/mL [68]
tient sam-
Amp For PSA
PF-4 &
& PSMA:
IL-6: For PSA
For PF-4 & PSMA:
& IL-6: Ab2/SA-HRP
SWCNF/Ab Ab2 /HRP (biphasic with better
(PCa
ples)patient IL-6PF-4 SWCNF/Ab1 1
For PF-4 & IL-6: For PF-4 & IL-6:
PF-4: 1–40 ng/mL PF-4:
IL-6: 1 ng/mL
0.03 ng/mL [68]
samples) sensitivity below 350
SWCNF/Ab1 Ab2 /SA-HRP IL-6: 50–500 pg/mL
pg/mL)
(biphasic with better
Amp PSAIL-6 sensitivity below
IL-6: 0.23
PSA: 0.03pg/mL
ng/mL
(PCa pa- 350 pg/mL)
GSH-AuNPs/Ab1 MP/Ab2/HRP – [15]
tient sam- IL-6 IL-6: 0.30 pg/mL
Amp(PCa PSA PSA: 0.23 pg/mL
ples)
patient GSH-AuNPs/Ab1 MP/Ab2 /HRP – [15]
samples)
Amp IL-6 PSA: 61 fg/mL–3.9 IL-6: 0.30 pg/mL
PSA PSA: 15 fg/mL
(PCa pa- PSA:pg/mL *
61 fg/mL–
PSA ERGO/Ab1 Ab2/Fe3O4 NPs/GO PSA: 15 fg/mL [119]
Amp(PCa
tient sam- 3.9 9.8
PSMA: pg/mL *
fg/mL–10
patient PSMA ERGO/Ab1 Ab2 /Fe3 O4 NPs/GO PSMA: 4.8 fg/mL [119]
ples) pg/mL *
PSMA: 9.8 fg/mL–
samples) PSMA PSMA: 4.8 fg/mL
PSA PSA:10 pg/mLng/mL
0.14–34.2 * PSA: 140 pg/mL
VEGF-D: 0.09–23.8
VEGF VEGF-D: 90 pg/mL
ng/mL
ERG: 0.015–3.9
ERG ERG: 15 pg/mL
ng/mL
IGF-1: 0.013–3.4
IGF-1 IGF-1: 13 pg/mL
ng/mL
Amp PDDA/GSH-AuNPs/Ab1 MP/Ab2-HRP CD-14: 0.13–32.5 [55]
IGFBP-3 CD-14: 130 pg/mL
ng/mL
IGFBP-3: 0.15–38.7
CD-14 IGFBP-3: 150 pg/mL
ng/mL
PEDF-1: 0.09–11.2
PEDF PEDF-1: 90 pg/mL
ng/mL
GOLM-1: 0.015–1.95
GOLM-1 GOLM-1: 15 pg/mL
ng/mL
Sensors 2021, 21, 5023 25 of 33

Table 3. Cont.

Sensor surface Linear Detection

Technique Biomarkers modification Detection Label Range Limit of Detection Ref

PSA PSA:
0.14–34.2 ng/mL PSA: 140 pg/mL

VEGF VEGF-D: VEGF-D:

0.09–23.8 ng/mL 90 pg/mL

ERG ERG:
0.015–3.9 ng/mL ERG: 15 pg/mL

IGF-1 IGF-1:
0.013–3.4 ng/mL IGF-1: 13 pg/mL
Amp PDDA/GSH- MP/Ab2 -HRP [55]
AuNPs/Ab1 CD-14:
IGFBP-3 0.13–32.5 ng/mL CD-14: 130 pg/mL

IGFBP-3: IGFBP-3:
CD-14 0.15–38.7 ng/mL 150 pg/mL
PEDF-1:
PEDF 0.09–11.2 ng/mL PEDF-1: 90 pg/mL

GOLM-1 GOLM-1: GOLM-1:

0.015–1.95 ng/mL 15 pg/mL
PSA: 2 pg/mL–
PSA
200 ng/mL *
PSMA: 0.15 pg/mL–
PSMA
SAM(MPA)/Ab1 MP/Ab2 /HRP 15 ng/mL * 0.05–2 pg/mL [123]
DPV
PF-4: 0.1 pg/mL–
PF-4
10 pg/mL *
IL-6: 0.05 pg/mL–
IL-6
5 ng/mL *
For VEGF & PSA:
DPV PSA Ab2 /PPLA NPs, where PSA: 1 ng/mL
(PCa patient GO/Apt Ab2 is a mixture of – [124]
samples) anti-VEGF and anti-PSA
VEGF antibodies VEGF: 50 pg/mL
SWV tPSA
(PCa patient – tPSA: 0.2 ng/mL
GO/AuNPs/Ab1 GO/AuNPs/Ab2 [125]
samples) fPSA fPSA: 0.07 ng/mL
PSA PSA: 0.1–10 ng/mL PSA: 0.1 ng/mL

PSMA Ab1 /MBs HRP/Ab2 /AuNRs PSMA: [69]

CV 0.8–400 ng/mL PSMA: 0.8 ng/mL

IL-6 IL-6: 5–1000 pg/mL IL-6: 0.005 ng/mL

fPSA SAM
EIS – – 1 ng/mL [129]
tPSA (MUA-2-ME)/Ab1
PSA PSA: 4 aM
SAM PSA glycans: down [130]
EIS PSA (MUA-MCH)/Ab1 SNA 4 a.m. to 40 nM
glycans to 4 a.m.
(~0.13 fg/mL)

PSA PSA:
EIS 0.64–62.5 ng/mL * PSA: 0.64 ng/mL
(PCa patient SAM(AUT)/MCH/
AuNPs/SAM(Apt-MCH) [131]
samples) PSA PSA glycans: PSA glycans:
glycans 0.26–62.5 ng/mL * 0.26 ng/mL
Abbreviations: 2-ME = 2-mercaptoethanol, Ab1 = capture antibody, Ab2 = detection antibody, Amp = Amperometry, Apt = DNA ap-
tamer, AuNPs = Gold nanoparticles, AuNRs = Gold nanorods, AUT = 11-amino-1-undecanothiol, CD-14 = Cluster of differentiation-14,
CV = Cyclic Voltammetry, DPV = Differential Pulse Voltammetry, EIS = Electrical Impedance Spectroscopy, ERG = Erythroblast transforma-
tion specific related gene, ERGO = electrochemically reduced graphene oxide, Fe3 O4 NPs = Iron oxide nanoparticles, fPSA = Free PSA,
GO = Graphene oxide, GOLM-1 = Golgi membrane protein-1, GSH = Glutathione, HRP = horseradish peroxidase, IGF-1 = Insulin-like
Growth Factor-1, IGFBP-3 = Insulin-like Growth Factor binding protein-3, IL-6 = Interleukin-6, MB = magnetic beads, MCH = 6-mercapto-
1-hexanol, MP = magnetic nanoparticles, MPA = mercaptopropionic acid, MUA = 11-mercaptoundecanoic acid, PDDA = poly(diallyl
dimethylammonium chloride), PEDF = Pigment epithelium-derived factor, PF-4 = Platelet factor-4, PPLA NPs = Poly-L-lactide nanopar-
ticles, PSA = Prostate-Specific Antigen, PSMA = Prostate-specific membrane antigen, SA = streptavidin, SAM = self-assembly mono-
layer, SNA = Sambucus nigra, SWCNF = single-wall carbon nanotube forests, SWV = Square Wave Voltammetry, tPSA = Total PSA,
VEGF = Vascular endothelial growth factor. * Dynamic detection ranges.
Sensors 2021, 21, 5023 26 of 33

3.3. Potential Companion Diagnostic Devices Using Integrated Biosensor Systems

Overall, the research prototypes discussed in this review have the potential to be
just as accurate as traditional protein detection methods, and they can be implemented in
conjunction with the ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and
Robust, Equipment-free, and Delivered to those who need it) strategy of companion diag-
nostics devices [132,133]. Thus, considerable attention has been dedicated to the creation of
cost-effective integrated biosensor systems, such as the application of microfluidic systems,
while also considering multiplexed detection techniques, as stated above. Microfluidic
system integration is a crucial technique to explore for clinical usage by end users, as it can
enable high-throughput detection while requiring smaller sample amounts, and possible
automated control [89,134]. In addition, microfluidic systems can incorporate important
steps performed in a clinical laboratory setting, such as sample preparation, molecular
recognition, and signal amplification procedures (so-called lab-on-chip devices), negating
the need for expensive specialist equipment or skilled professionals [135].
Key fabrication technologies used to detect PCa protein biomarkers included paper-
based techniques, polymer (plastic-based) microfabrication, and the usage of microar-
rays [118]. When incorporating multiplexing approaches to either optical or electrochemical
detection methods, each fabrication technology has some limitations, which can have an im-
pact on translating prototypes into clinical utility that is appropriate for end users [89,136].
Because electrical instrumentation is difficult to incorporate, paper-based techniques such
as LFIAs are more compatible with optical measurements. Although polymer (plastic-
based) microfabrication designs are more efficient for electrochemical detection approaches,
such platforms are not mass-produced in terms of electronics integration. As a result,
the time and expense required to complete such processes significantly escalates [136].
Furthermore, increasing the number of spatially separated detection sites or regions for
PCa protein biomarkers necessitates the inclusion of additional components such as more
controls, valves, and detection/capture chambers, resulting in a complicated fabrication
process. On the other hand, simple designs without controlled sites or regions could lead to
cross-reactivity of sample solutions introduced to the biosensor system, resulting in unreli-
able detection results. Meanwhile, when using sandwich immunocomplexes as the surface
chemistry, the inclusion of additional labels will necessitate additional washing steps in
between incubation steps and/or detection measurements. Label-free surface chemistries,
on the other hand, may impair detection sensitivity for certain PCa protein biomarkers
with low serum concentrations. As a result, a compromise must be established between the
integrated biosensor system’s complexity and the multiplexed detection approach [20,89].
Due to being mass-produced internationally at a cheap cost and being compact/portable,
printed circuit boards (PCBs) could alleviate some of the issues faced with plastic-based
biosensors for electrochemical biosensor systems [136,137]. Furthermore, PCBs have long
been integrated with both electronic and microfluidic devices, requiring low sample vol-
umes and coinciding with the ASSURED approach to xPOCT (lab-on-PCB approach) [136].
Therefore, multiplexing techniques combined with PCBs can potentially help secure consis-
tent clinical outcomes for PCa patients that are highly sensitive and specific.

4. Conclusions and Future Perspectives

We summarized some of the emerging PCa protein biomarkers reported to date
that may be relevant for PCa diagnosis, prognosis, and treatment monitoring, in con-
junction with the widely known biomarker PSA. Although several markers have been
recognized, these are still yet to be validated as PCa biomarkers [138]. In addition, this re-
view looked at recent developments in miniaturized biosensor systems using optical and
electrochemical detection techniques, to detect multiple PCa protein biomarkers simultane-
ously. Such miniaturized biosensor surfaces have been modified using a variety of labels
or nanomaterials, such as enzymes, metallic nanoparticles, or graphene sheets, in order to
achieve systems with multiplexing capacities. In addition, some of the biosensor platforms
mentioned have been integrated with a microfluidic system, allowing the implementa-
Sensors 2021, 21, 5023 27 of 33

tion of complex functions as seen in clinical laboratories (lab-on-a-chip approach). Thus,

effectively achieving sensitive results while using smaller reagent and sample volumes
in a reliable and accurate manner that is comparable to conventional clinical laboratory
performance [139]. Additionally, the manufacture of 3D-printing microfluidic systems
provides cost-effective and simple fabrication processes compared to conventional meth-
ods [52,99,108,139]. Moreover, analyzed results were well correlated with conventional
methods, such as ELISA single-analyte kits. However, more work is needed to trans-
late biosensor detection platforms into commercially available xPOCT devices in order
to deliver highly cost-effective and sensitive results that leads to better management of
PCa treatment in near-patient settings in a timely manner [20]. This would allow the use
of multiplexed companion diagnostic devices as alternatives to conventional PCa diag-
nostic techniques, such as DRE, transrectal ultrasonography (TRUS), positron emission
tomography (PET) and/or magnetic resonance imaging (MRI) [24].
Overall, recent developments showed that modifications to the surface of the biosen-
sor can provide efficient and sensitive multiplexed protein detection. However, further
research is needed to achieve simpler modification strategies that can still produce ultra-
sensitive and clinically relevant detection of serum PCa-associated protein biomarkers.
Increased availability of commercial multiplexed companion diagnostic devices could
provide portable xPOCT devices in near-patient settings. For instance, bench-top analyzers
or handheld devices that do not require highly trained professionals could be used within
a primary clinical setting, e.g., a hospital or a GP clinic. If the established handheld de-
vices are patient-friendly, it could be beneficial for both primary healthcare professionals,
but also for patients to use at home [140]. Thus, developing xPOCT devices that achieve
sensitive and specific results using a minute serum sample, and are comparable to the
results evaluated in clinical laboratories using conventional single-analyte protein detec-
tion kits such as ELISA [20,141]. Moreover, efforts have been made to integrate wireless
networks with xPOCT devices in order to effectively communicate and transfer real-time
and highly sensitive results, aiding to prevent unnecessary misdiagnosis and unnecessary
treatment [20,90,142]. Current research developments are focused on the detection of
multiple proteins biomarkers in relation to early PCa screening and diagnosis. However,
there is a greater need in the future for multiplexed PCa companion diagnostic devices to
aid clinicians who need to make a conclusive decision on the ideal treatment pathway to
be considered. Therefore, the provision of highly personalized approaches to PCa treat-
ment management, particularly during key diagnostic, active surveillance or monitoring
milestones while PCa patients are undergoing treatment, is in great need [21,140].
Many different methods have been identified in this review that could lead to mul-
tiplexed systems, some with extremely low limits of detection and/or high selectivity.
However, each system would have advantages and drawbacks, and which would make
it to market depends mostly on the clinical application: test by healthcare professional
versus test at home, clinical ranges required, diagnostic or monitoring, number of proteins
required to be measured, sample volumes available, cost, etc. It should also be noted that
although we focused on protein biomarkers, a range of other biomarkers are of interest
for PCa diagnosis and, in particular PCa prognosis such as microRNAs, circulating tumor
DNA (ctDNA), circulating tumor cells (CTCs), etc. Several of the systems described could
integrate true multiplexing capabilities by measuring different types of biomarkers.

Author Contributions: Writing—original draft preparation, J.A.-B.; writing—review and editing,

P.E.; supervision, D.M. and P.E. All authors have read and agreed to the published version of
the manuscript.
Funding: J.A.-B. was funded by a University of Bath Research Studentship.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Sensors 2021, 21, 5023 28 of 33

Conflicts of Interest: The authors declare no conflict of interest.

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