Biosensors and Bioelectronics 240 (2023) 115644

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Biosensors and Bioelectronics

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Current progress, strategy, and prospects of PD-1/PDL-1 immune

checkpoint biosensing platforms for cancer diagnostics, therapy
monitoring, and drug screening
Katarzyna Ratajczak 1, Hubert Grel 1, Piotr Olejnik 1, Slawomir Jakiela **, Magdalena Stobiecka *
Department of Physics and Biophysics, Warsaw University of Life Sciences (SGGW), 159 Nowoursynowska Street, 02776, Warsaw, Poland

A R T I C L E I N F O A B S T R A C T

Keywords: Recent technological advancements in testing and monitoring instrumentation have greatly contributed to the
Optical biosensors progress in cancer treatment by surgical, chemotherapeutic and radiotherapeutic interventions. However, the
Surface plasmon resonance mortality rate still remains high, calling for the development of new treatment strategies with higher efficacy.
Microfluidic systems
Extensive efforts driven in this direction have included broadening of early cancer screening and applying
PD-1/PD-L1 pathway
Immune checkpoint inhibitors screening
innovative theranostic nanotechnologies. They have been supported by platforms introduced to enable the
Exosomal PD-L1 detection and monitoring of cancer biomarkers, inhibitors, and other agents, able to slow down cancer pro­
gression and prevent metastasis. Despite of the well-recognized principles of the immune checkpoint blockade,
the efficacy of immunotherapy achieved so far does not meet the well-founded expectations. For a successful
cancer treatment, highly sensitive, robust, and inexpensive multiplex biosensors have to be designed to aid in the
biomarkers monitoring and in the development of new inhibitors. In this review, we provide an overview of the
efforts undertaken to aid in the development and monitoring of anticancer immunotherapy, based on the pro­
grammed cell-death immune checkpoint (PD-1/PDL-1) blockade, by designing biosensors for the detection of
relevant cancer biomarkers and their inhibitors screening. This review also emphasizes alternative targets made
by exosomes carrying PD-L1 overexpressed in cancer cells and passed into the excreted exosomes. Evaluated are
also novel targeted drug delivery nanocarriers, providing simultaneous biosensing, thereby contributing to the
emerging immune checkpoint cancer therapy. On the basis of the current trends and the emerging technologies,
future perspectives of cancer diagnostics and treatment monitoring using biosensing platforms are projected.

1. Introduction well recognized immune checkpoint mechanisms (Dong et al., 1999;

Considerable progress has recently been achieved in cancer treat­
ment, with advancements in precision surgical techniques, nanotech­
nology driven targeted chemotherapy, and multi-modal radiotherapy.
These advancements have strongly been supported by novel diagnostics
and monitoring technologies enabling largely successful management of
cancer growth retardation and prevention of metastasis (Corthay, 2014;
Couzin-Frankel, 2013; Farkona et al., 2016; Janssen et al., 2017; Kauf­
man et al., 2019; Weigmann, 2016). However, the use of less invasive
treatments involving the organism’s own defenses against cancer, based
on the immune system ability to selectively kill cancer cells, has only
been at the beginning stages of development, despite of the generally
Garrett and Collins, 2011; Pardoll, 2012). Since the attempts at the
application of cancer immunotherapy have been met with varying de­
gree of success, the great potential of immune checkpoint controlling
has yet to be realized (Brahmer et al., 2012; Goodman et al., 2017; Ribas,
2012). Due to the projections of the continuing growth in the number of
diagnosed cancer cases (2020; Siegel et al., 2021) (Fig. 1A), extensive
efforts of scientists and clinicians have been focused on broadening of
early cancer screening and on the development of new therapeutic ap­
proaches, including, among others, the induced immunotherapies
against cancer. In this review, we focus on cancer immunotherapies,
with particular attention paid to the progress in the development of
methods and biosensing devices for monitoring of biomarkers involved

* Corresponding author.
** Corresponding author.
E-mail addresses: slawomir_jakiela@sggw.edu.pl (S. Jakiela), magdalena_stobiecka@sggw.edu.pl (M. Stobiecka).
1
Katarzyna Ratajczak, Hubert Grel and Piotr Olejnik contributed equally to this work.

https://doi.org/10.1016/j.bios.2023.115644
Received 25 July 2023; Received in revised form 22 August 2023; Accepted 26 August 2023
Available online 28 August 2023
0956-5663/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
K. Ratajczak et al.
in the immune checkpoint controlling, such as the programmed cell
death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), in­
hibitors, and moderators of immune checkpoints, as well as the drug
molecules under development. Hence, we aim in this review at evalu­
ating novel platforms for monitoring of biomarkers and immunodrugs,
such as the biosensors arrays, microfluidic devices, optical fiber grat­
ings, functionalized gold nanoparticles and nano-shells, gated resonance
energy transfer (gRET) plasmonic nanoprobes and other biosensing
devices.
Among various modalities of cancer treatment, the immunotherapy
is by far the most natural one as it utilizes the components of the or­
ganism’s own immune system to fight and eradicate cancer cells (Asa­
dujjaman et al., 2020; Couzin-Frankel, 2013; Farkona et al., 2016;
Kaufman et al., 2019; Xia et al., 2019). Immunotherapy complements
commonly applied but highly invasive cancer treatments, including
surgery, radiotherapy, and conventional chemotherapy. Due to the key
features of immune-mediated therapy, which include biocompatibility,
specificity, breadth of response, and memory (Kaufman et al., 2019),
enabling noninvasive cancer treatment with no collateral damage to
healthy cells, it is becoming a powerful clinical strategy against cancer.
The group of immunotherapies consists of five distinct classes that
include: (i) immunomodulators; (ii) cell-based immunotherapies (e.g.
chimeric antigen receptor T cells (CAR T)); (iii) antibody-based targeted
therapies that co-stimulate cells or block the checkpoint pathways (e.g.
PD-1/PD-L1); (iv) cancer vaccines; and (v) oncolytic viruses, designed to
selectively infect and kill cancer cells, while inducing systemic
anti-tumor immune response and memory (García-Aranda and
Redondo, 2019; Gong et al., 2019; Iwai et al., 2002; Marshall and
Djamgoz, 2018; Ribas, 2012; Xie et al., 2018) (Fig. 1B).
The immune system is able to recognize most of the cancer cells
(Corthay, 2014; Gonzalez et al., 2018; Janssen et al., 2017; Pandya et al.,
2016). Unfortunately, some cancer cells can evade the immune system
and grow (Marshall and Djamgoz, 2018). It has been found that the
prevailing mechanism of immune system evasion involves activation of
the checkpoints of the programmed cell death (PD) pathway (Han et al.,
2020).
PD-1, also known as PDCD1 or CD279 (cluster of differentiation No.
279), is highly expressed in the membrane of activated T cells, B cells,
and natural killer cells and its main role is to regulate the immune
response by suppressing inflammatory activity. PD-1 is a 50–55-kDa
type I transmembrane glycoprotein (Wu et al., 2019a). The ligand
interacting with PD-1, called PD-L1, known also as CD274 or B7-H1, is a
transmembrane protein which is expressed in tumor cells, macrophages,
mast, or dendritic cells. Both PD-1 and PD-L1 belong to the immune
checkpoint protein family. The combination of PD-L1 and PD-1 can
inhibit the activation, proliferation and anti-tumor function of CD8+ T
cells, thus enabling the tumor immune escape (Ai et al., 2020).
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Biosensors and Bioelectronics 240 (2023) 115644
A low-level expression of PD-L1 has been encountered in many cell
types. However, an elevated expression of PD-L1 on the surface of tumor
cells has generally been observed and associated with worse prognosis
for many tumors (Mahoney et al., 2015). PD-L1 causes the inhibition of
kinase signaling pathways upon binding to the immune cell PD-1 re­
ceptors, resulting in suppressing of the T cells activity and taming of
immune response (Aguiar et al., 2017; Davis and Patel, 2019; Incorvaia
et al., 2019; Patel and Kurzrock, 2015). In this way, cancer cells
expressing PD-L1 can easily evade immune reaction of the host and grow
freely by binding T cells (Santarpia and Karachaliou, 2015).
The crystal structure of human PD-1/human PD-L1 complex was
recently reported by Zak et al., (2015). The Authors have shown mo­
lecular details of the human PD-1/PD-L1 interaction based on the X-ray
structure of the complex. They have identified two spots, among three
major active spots, which may facilitate the formation of a druggable
pharmacophore capable of disrupting the PD-1/PD-L1 interaction.
The mechanisms of the immune activity control have been eluci­
dated upon the discovery of immune checkpoints, leading to the
development of anticancer immunotherapy concept, recognized in 2018
with a Nobel Prize in Physiology or Medicine, awarded to James Allison
and Tasuku Honjo (Huang and Chang, 2019; Zang, 2018). Honjo and his
group discovered, on T cells, the programmed death molecule-1 (PD-1),
a member of the immunoglobulin superfamily (Ishida et al., 1992),
while Allison and his colleagues have shown that the monoclonal anti­
body against the immunosuppressive molecule, cytotoxic T-lymphocy­
te-associated protein 4 (CTLA-4), blocked the CTLA-4 function and
enhanced the anti-tumor immunity. The mice treated with anti-CTLA-4
antibodies completely rejected their tumors (Leach et al., 1996).
The excellent recent reviews related to the PD-1/PD-L1 cancer
immunotherapy have mainly focused on the mechanisms involving
PD1/PD-L1 checkpoint inhibitors and their applications in tumor
immunotherapy (Doroshow et al., 2021; Liu et al., 2021), the develop­
ment of biophysical and biochemical assays for binding affinity mea­
surements to identify small molecule inhibitors against PD-1/PD-L1 (Liu
et al., 2021), as well as the meta-analysis of predictive biomarkers for
immune checkpoint inhibitor (ICI) therapy (Mariam et al., 2023). In this
review, we focus on the evaluation of the progress in the development of
two classes of monoclonal antibodies (anti-PD-1 and anti-PD-L1) and the
aptamers-based biosensors for the detection of the programmed cell
death protein-1 (PD-1), PD-L1 ligand, and biomolecules able to inhibit
or moderate the immune checkpoint proteins, with the emphasis on the
invaluable aid of a variety of different biosensing platforms, including
microfluidic devices, biosensors, fiber gratings coated with gold
nano-shells, as well as plasmonic nanoprobes based on gated resonance
energy transfer (gRET) for the cancer diagnosis and treatment. The
sensing systems enabling PD-1/PD-L1 inhibitors’ screening are reviewed
as well. The efficacy of PD-1/PD-L1 inhibitors, being the key acting
Fig. 1. (A) Estimated global burden of cancer in 2018
and 2040 according to World Health Organization
(WHO) (2020) (Licence: CC BY-NC-SA 3.0 IGO); (B)
Classes of immunotherapies. PD-L1 mAb, pro­
grammed cell death ligand 1 monoclonal antibody;
PD-1 mAb, programmed cell death 1 monoclonal
antibody; CTLA4 mAb, monoclonal antibodies tar­
geting cytotoxic T-lymphocyte antigen- 4; IL-2,
interleukin-2; IFN-α, interferon -α; IL-15, inter­
leukin-15; IL-21, interleukin-21; T-Vec, talimogene
laherparepvec; TCR, T cell receptor; CAR T, chimeric
antigen receptor T cells.
K. Ratajczak et al.
elements in the cancer immunotherapy, has been evaluated based on
recent investigations utilizing surface plasmon resonance (SPR) and
homogeneous time-resolved fluorescence (HTRF) techniques. We also
assess the utility of new theranostic drug nanocarriers, such as the poly
(lactic-co-glycolic acid) (PLGA) nanoparticles, micelles with functional
penetrating peptides conjugated with cholesterol and histidine, and
apoferritin nanocages, as the carriers of small interfering RNAs (siRNAs)
for inhibition of PD-L1/PD-1 expression. The potential and challenges of
utilizing PD-L exosomes as the novel therapeutic target are also high­
lighted. Furthermore, we discuss the future outlook and potential ap­
plications of biosensors and exosomes for testing the immune
checkpoint blockade, drug development, and cancer treatment. Finally,
we evaluate the prospects of innovative targets for cancer diagnostics
and immunotherapy.
2. PD-1/PD-L1 immune checkpoint blockade
The development of cancer treatments able to induce an efficient
immune checkpoint blockade by binding PD-1 or PD-L1, is urgently
needed to successfully treat cancer. To interrupt the negative control of
immune response, the brakes of immune checkpoints (i.e., the bridges
PD-1/PD-L1) must be released. While any compound with strong affinity
to PD-1, able to suppress immune-controlling activity of PD-1, can in
principle be considered as the potential candidate for a checkpoint in­
hibitor, two classes of monoclonal antibodies (MoAbs), and have been
designed to specifically serve for this purpose: the anti-PD-1 and the
anti-PD-L1 MoAbs. The antibodies against PD-1 are fully human and
humanized IgG4 and the antibodies against PD-L1 are IgG1 isotypes
with genetically modified Fc fragments (Homet Moreno and Ribas,
2015; Passiglia et al., 2016).
To better understand the scope and challenges of controlling the
immune checkpoints, for the sake of designing biosensing devices
capable of monitoring the activity of key biomolecules and designer
drugs involved in checkpoint operation, we need to consider first the
biomedical principles of this autonomous checkpoint functioning. The
comprehensive background provided in this Section enables better un­
derstanding of the modifying power, the proteins PD-1, PD-L1, and the
inhibitor drugs have on the tumor survivability.
The immune checkpoint inhibitors prevent binding of T cells to
cancer cells (i.e. binding of PD-1 receptors to PD-L1 ligands), and thus,
block the immune checkpoints and enable an uninterrupted release of
immune attack on cancer cells resulting in the elimination of cancer
(Riley et al., 2019; Wu et al., 2019b). Anti-PD-1 antibody binds to PD-1
and anti-PD-L1 binds to PD-L1 preventing the binding of PD-1 to its li­
gands and PD-L1 to PD-1, releasing the tumor-specific killing ability of T
cells. Therefore, the introduction of immune checkpoint inhibitors has
recently become a promising therapy for cancer treatment (Akinleye and
Rasool, 2019; Fritz and Lenardo, 2019; Garrett and Collins, 2011; Li
et al., 2016; Lipson et al., 2015; Pardoll, 2012; Wu et al., 2019a; Xu et al.,
2018). The blockade of checkpoints by antibodies against PD-1 and
PD-L1 was investigated for cohorts of a large number of patients with
melanoma (Bhandaru and Rotte, 2017; Hu-Lieskovan and Ribas, 2017),
non-small-cell lung carcinoma (NSCLC) (Aguiar et al., 2017; Passiglia
et al., 2016), renal cell carcinoma (RCC), bladder cancer (BC), Hodgkin
Lymphoma, triple-negative breast cancer (TNBC), and hepatocellular
carcinoma (HCC), as well as for hematologic malignancies (Brahmer
et al., 2012; Homet Moreno and Ribas, 2015; Macek Jilkova et al., 2019;
Philips and Atkins, 2014; Villasboas and Ansell, 2016; Xu et al., 2018).
To improve the clinical outcomes for patients, the combination
therapies utilizing a set of different immune checkpoint inhibitors are
also frequently applied. The new emerging immune checkpoint target
for immunotherapy consists of a T cell immunoreceptor with Ig and
immunoreceptor tyrosine-based inhibitory motif (ITIM) domains
(TIGIT), mainly expressed on natural killer (NK) cells and regulatory T
cells (Tregs) (Zhao et al., 2023; Harjunpää and Guillerey, 2019; Dougall
et al., 2017). In the first, in-human phase 1 study of patients with
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Biosensors and Bioelectronics 240 (2023) 115644
advanced solid tumors (NCT02964013), Niu et al., (2022) have found
that the humanized immunoglobulin G1 monoclonal antibody, that
binds to TIGIT vibostolimab (MK-7684), with an anti-PD-1 antibody
pembrolizumab, was well tolerated, and demonstrated antitumor ac­
tivity in patients with non-small-cell lung cancer (NSCLC). Thibaudin
et al., (2022) have suggested that the combination of atezolizumab
(anti-PD-L1) and tiragolumab (anti-TIGIT) in ex vivo evaluation could
restore function of CD4 and CD8 tumor-infiltrating lymphocytes (TILs)
in microsatellite stable colorectal cancers (MSS-CRC). Hung et al.,
(2018) have examined the efficacy and mechanism of dual anti-PD-1
and anti-TIGIT checkpoint blockade in a murine glioma model.
Another immune checkpoint utilized as the target in combination cancer
immunotherapy is the type I transmembrane glycoprotein, member of
the immunoglobulin (Ig) superfamily, that interacts with nectin and
nectin-like proteins, CD96 (TACTILE) (Feng et al., 2023; Liu et al.,
2020). Mittal et al., (2019), tested the anti–PD-1/CD96/TIGIT in a
triple-combination therapy. They have demonstrated that this triple
combination is superior in reducing the tumor growth and improving
the survival of mice in B16F10 melanoma and CT26 colon carcinoma,
compared with monotherapy or dual-combination therapy. The combi­
nation of antibodies against CD96 and PD-1 has also enhanced the delay
of tumor growth and prolongation of the survival of tumor-bearing mice
(TC-1 tumor-bearing C57BL/6 mouse) (Wang et al., 2022). In a synge­
neic model of prostate cancer that is resistant to immune checkpoint
inhibitors (ICIs), inhibition of the binding of vascular endothelial growth
factor (VEGF) to neuropilin-2 (NRP2) using a mouse-specific anti-NRP2
monoclonal antibody (mAb) resulted in necrosis and tumor regression
compared with both an anti–PD-L1 mAb and control immunoglobulin G
(Wang et al., 2023). However, de Filette et al. (de Filette et al., 2019) in a
systematic analysis have listed adverse events provoked by single agent
checkpoint blockade, further reinforced by combined treatment. Patel
et al., (2022) have investigated the role of regulatory B (Breg) cells in the
development of severe immune-related adverse events in lung cancer
patients treated with anti-PD-1/PD-L1.
The immune checkpoint inhibitors, based on monoclonal antibodies
against PD-1 receptor, such as nivolumab and pembrolizumab, and
those based on the anti-PD-L1 antibodies, such as atezolizumab, avelu­
mab and durvalumab, were successfully approved by the US Food and
Drug Administration (FDA) for different types of cancer and commer­
cialized over the last few years (Homet Moreno and Ribas, 2015;
Novotny et al., 2016; Wu et al., 2019a; Xu et al., 2018). According to the
Web of Science website (https://www.webofscience.com/wos/woscc/­
citation-report/5dddc8d6-e2f9-4c2b-a7a2-62e9a7416f54-959ad3d6
2023), the publications record concerning the PD-1/PD-L1 immune
checkpoint inhibitors began to increase from 2014 after the first
approval of PD-1/PD-L1 inhibitor and the publications growth rate is
still increasing (Fig. 2A). It also indicates that the number of research
groups developing the biosensors for detection of PD-1/PD-L1 immune
checkpoint proteins is only beginning to increase and more de­
velopments will be published in near future. In Fig. 2 B and Table 1, we
summarized the PD-1/PD-L1 inhibitor drugs approved recently, along
with the information on targeted cancer, date of approval, and adverse
side events.
3. Biosensing technologies for the detection of PD-1/PD-L1
immune checkpoint proteins
To provide a simple means for monitoring of immune checkpoint
activity, many research groups have recently been actively working on
the development of new analytical methods and biosensing technologies
for fast and sensitive determination of checkpoint proteins (Briones
et al., 2020; Chou et al., 2018; Reza et al., 2019; Wuethrich et al., 2019).
As will be discussed in this section, for the detection of PD-1 and PD-L1
proteins, the microfluidic biosensing platforms, immunosensors, apta­
sensors, as well as plasmonic assays, have recently been designed. The
Authors have utilized various techniques, including flow cytometry,
K. Ratajczak et al. Biosensors and Bioelectronics 240 (2023) 115644

Fig. 2. (A) The records for PD-1/PD-L1 immune checkpoint inhibitors topic according to WoS website; (B) Examples of immune checkpoint inhibitors approved
by FDA.

Table 1
Approved PD-1/PD-L1 inhibitors.
Immune Approved drugs Cancer treatment Approval Treatment-related adverse events Ref.
checkpoint date
target

PD-1 nivolumab melanoma 2014 hypothyroidism, hyperthyroidis, thyroiditis, (Bhandaru and Rotte, 2017;
non-small cell lung cancer 2015 hypophysitis, diabetes mellitus, primary adrenal de Filette et al., 2019;
(NSCLC) insufficiency, Ksienski et al., 2019)
metastatic renal cell carcinoma 2015 dermatitis, colitis
Hodgkin lymphoma 2016
recurrent and metastatic 2016
squamous cell carcinoma of
head and neck (SCCHN)
urothelial carcinoma 2017
pembrolizumab metastatic small cell lung 2018 hypothyroidism, hyperthyroidis, thyroiditis, (de Filette et al., 2019;
cancer hypophysitis, diabetes mellitus, primary adrenal Ksienski et al., 2019;
melanoma 2014 insufficiency, pneumonitis, athralgis, dermatitis Passiglia et al., 2016)
non-squamous non-small cell 2015
lung cancer (NSCLC)
non-muscle invasive bladder 2020
cancer (NMIBC)
cemiplimab-rwlc advanced cutaneous squamous 2018 diarrhea, arthralgia, hypothyroidism, muscle (Ahmed et al., 2019)
cell carcinoma (CSCC) weakness, macularpapular rash, nausea, pruritis,
rash, cough
PD-L1 atezolizumab urothelial carcinoma 2016 hypothyroidism, diabetes mellitus (de Filette et al., 2019)
metastatic non-small cell lung 2016
cancer (NSCLC)
durvalumb locally advanced or metastatic 2017 hypothyroidism (de Filette et al., 2019)
urothelial carcinoma
avelumab Merkel-cell carcinoma 2017 hypothyroidism, hyperthyroidis, diabetes mellitus, (de Filette et al., 2019)
primary adrenal insufficiency
Combination pembrolizumab plus advanced endometrial 2019 hypertension, diarrhea, fatigue, decreased appetite, (Arora et al., 2020; Makker
thearpy lenvatinib carcinoma hypothyroidism, nausea, stomatitis, pain and et al., 2020)
arthralgia, dysphonia
Combination ipilimumab plus irresectable metastatic 2015 hypothyroidism, hyperthyroidis, thyroiditis, (Bhandaru and Rotte, 2017;
therapy nivolumab melanoma hypophysitis, primary adrenal insufficiency, colitis, de Filette et al., 2019)
liver abnormalities, myocarditis

surface enhanced resonance spectroscopy, fluorescence, and electro­
chemistry for analytical signal measurements (Table 2).
The advantages and disadvantages of different biosensing technol­
ogies are discussed in detail in subsections 3.1-3.3 and they are also
summarized in Table 2. Various detection methods used in these tech­
nologies and the achieved detection limits are also evaluated in this
Table.
3.1. Integrated microfluidic systems for immune checkpoint biomarkers
detection
The microfluidic devices have demonstrated good sensitivities for
analyzed antigens and significant advantages, such as the system mini­
aturization, simplicity, automation, minimal volume of samples and
reagents, as well as multiple working channels enabling simultaneous
measurements for multiple biomarkers detection.
Reza et al., (2019) have developed an integrated surface-enhanced
Raman scattering (SERS)- microfluidic platform, with an
alternating-current electrohydrodynamic (ac-EHD, f = 1.1 kHz and Vpp
= 120 mV) mass transport, for the detection of immune checkpoint
proteins (Fig. 3). The gold electrodes were functionalized with graphene
oxide (GO) and hemagglutinin (HA) antibodies using 1-ethyl-3-(3-dime­
thylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS)
chemistry (EDC: 0.2 M; NHS: 0.05 M; mixing volume ratio of 1: 1). Next,
the nanoyeast single chain variable fragments (NYscFv), specific to
immune checkpoints PD-1, PD-L1, and LAG-3, tagged with human
influenza hemagglutinin antigen (10 μg/mL), were attached. Owing to
laborious, long and costly production times of antibody affinity

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K. Ratajczak et al. Biosensors and Bioelectronics 240 (2023) 115644

Table 2
Biosensing technologies for detecting PD-1/PD-L1 immune checkpoint proteins.
Biosensor Immune Detection method Limit of Real Advantage(s) Disadvantage(s)/challenge(s)/ Ref.
check- detection sample limits of detection
point

Immuno-sensor sPD-L1 surface plasmon resonance 1.75 ng serum sensitive and low concentration in serum and (Hu et al., 2021)
(SPR); /mL samples selective; various body fluids;
cyclic voltammetry (CV); alternative for anti- natural SIBP obtained without
electrochemical impedance bodies; tedious selecting process;
spectroscopy (EIS) strong binding ability tested in diluted serum from
healthy donors and cancer patients
Immuno-sensor PD-L1 surface plasmon resonance 3.29 ng/ human high surface-to- dual recognition; (Huang et al.,
(SPR); mL plasma volume ratio; tested in blood samples collected 2021)
cyclic voltammetry (CV); strong magneto- from healthy donors and cancer
electrochemical impedance optical effect; patients
spectroscopy (EIS); high sensitivity;
atomic force microscope low detection limit;
(AFM) specificity;
template-free method;
excellent electrical
conductivity
Aptasensor PD-L1 flow cytometry 10 cells/ n.a. highly affinite and not tested with real clinical (Yazdian-Robati
mL selective; samples; et al., 2017)
alternative for small population of cancer cells;
antibodies internalization at 37 ◦ C is better
than at 4 ◦ C;
require multiple washing steps
Nanoplasmo-nic PD-L1 gated fluorescence 1.2 nM n.a. very sensitive; not tested with real clinical (Grel et al., 2020)
assay resonance energy transfer simple fabrication; samples;
(gRET) rapid detection could only detect one biomarker;
require modification by antibodies
to increase the specificity of the
system
ExTFG-LSPR sPD-L1 surface plasmon resonance 1 pg/mL PBS probing processes decreased in senitivity when (Luo et al., 2019)
immunosen-sors (SPR) (0.04pM) within the nanometer applied in FBS sample;
5 pg/mL fetal scale; not tested with real clinical
bovine no effect of tempe- samples; detecting of small
serum rature on the sensor biomolecules (sPD-L1, 26 kDa); the
(FBS) response; detection range could be improved
stability of ExTFG; by using graphene oxide (GO) to
extremely specific encapsulate the gold nanoshells
rapid and label-free
detection;
160 nm gold nano-
shells have larger
specific surface area
than 80 nm
Chip-based array PD-1 SERS-microfluidic platform 100 fg/ spiked enable simultaneous complex fabrication process; (Reza et al.,
mL human detection; background removal from the raw 2019)
PD-L1 50 fg/mL serum rapid detection; spectra;
LAG-3 100 fg/ samples multiplexed facilitate simultaneous monitoring
mL detection; and suggest effective therapy
highly sensitive and
specific
alternative for anti-
bodies;
good reproducibility;
excellent storage
stability
sandwich-type sPD-1 SERS platform 6.17 pg/ spiked enable specific straightforward and (Li et al., 2018)
SERS mL human multiplexed environmentally friendly synthesis
platform sPD-L1 0.68 pg/ serum detection; workflow does not require
mL cheap and specific surfactants, high temperature or an
sEGFR 69.86 pg/ detection; organic phase;
mL alternative for anti- developed nanoyeast-scFvs in
bodies fragments of yeast cell walls;
not tested with real clinical
samples
microfluidic PD-L1 flow-proteometric (FAP) 30.5 pM HeLa cells rapid detection; combination a microfluidic-based (Chou et al.,
immunoassay microfluidic platform 2.33 pM MDA-MB- sensitive; system with fluorescence 2018)
231 cells standard-free tech- correlation spectroscopy (FCS) as
patient nique the platform FAP to automatically
breast determine;
tumor for the protein concentrations
tissue <0.13 pM extending the detection
samples time and increasing the flow speed
is necessary;
(continued on next page)

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K. Ratajczak et al. Biosensors and Bioelectronics 240 (2023) 115644

Table 2 (continued )
Biosensor Immune Detection method Limit of Real Advantage(s) Disadvantage(s)/challenge(s)/ Ref.
check- detection sample limits of detection
point

non-specific binding of antibody to

cell or tissue sample may limited
the sensitivity
microfluidic PD-1 alternating-current 5 pg/mL n.a. alternative for anti- not tested with real clinical (Wuethrich et al.,
hemagglutinin PD-L1 electrohydrodynamic (ac- 5 pg/mL bodies; samples; 2019)
(HA) LAG-3 EHD) nanofluidic read-out 50 pg/mL enable multiplexed ac-EHD system generates a
immunosen-sor detection from small nanoscopic fluid flow;
sample volumes; slow mass transport of the analytes,
simultaneous analysis causes slow response time and
of 28 samples; nonspecific interference from the
colorimetric read-out nontarget molecules

Fig. 3. (A) Schematic illustration of the SERS GO ac-EHD immunoassay for immmuno checkpoint blockade biomarkers. (B) Typical SERS spectra and dynamic range
for PD-1. The serum samples spiked with targets were run through the device under an ac-EHD field (f = 1.1 kHz and Vpp = 120 mV). Control experiments were
performed using blank serum samples (0 fg/mL). Adopted from (Reza et al., 2019).

reagents, the Authors have utilized recombinant affinity reagents
NYscFv, that can be generated quickly, at a low cost, with high speci­
ficity toward their target antigens and high stability (Grewal et al.,
2015). NYscFv designed as the new diagnostic protein capture agent and
alternative to full length monoclonal antibodies were generated by
mechanical fragmentation, followed by filtration to produce yeast-cell
wall fragments of varying sizes, with the single-chain variable frag­
ments (scFvs) complex remaining intact (Grewal et al. 2015, 2016).
Then, the samples (20 μL) containing immune checkpoint antigens
(PD-1 (100 fg/mL to 1 ng/mL), PD-L1 (50 fg/mL to 100 pg/mL), and
LAG-3 (100 fg/mL to 1 ng/mL) were injected into different channels of
the ac-EHD device. Subsequently, the captured antigens were detected
using three different SERS nanotags consisting of gold nanoparticles and
Raman reporters, including mercaptobenzoic acid (MBA, 5 μL of 1 mM)
for PD-1, 5,5′-dithio-bis-[2-nitrobenzoic acid] (DTNB, 10 μL of 1 mM) for
PD-L1, and 7-mercapto-4-methylcoumarin (MMC) for LAG-3. The
channel without the NYscFv was used as a negative control. The Authors
have shown that this SERS platform can be used for the detection of
immune checkpoint markers spiked in human serum down to LOD =
100 fg/mL. Thus, due to the sensitive multiplex detection of targets in
human serum samples, this method offers a high potential for fast cancer
screening. Also, the successful application of NYscFv in a biosensing
platform provides a promising low-cost alternative in comparison to the
expensive monoclonal antibodies used in current methods such as the
ELISA.
Biosensing technologies based on integrated microfluidic platforms
have also been developed for the determination of PD-L1 protein.
Recently, Chou et al., (2018) designed a flow-proteometric microfluidic
platform for analyzing proteins (FAP). The concentration of PD-L1 was
determined in vitro in cultured cancer cells (HeLa and MDA-MB-231)

6
K. Ratajczak et al.
and in vivo in breast tumor tissue samples from patients. The cells were
labeled first with Alexa Fluor 647-conjugated anti-PD-L1 antibody and
then the cell lysate was collected and injected into the FAP platform for
PD-L1 detection. The Authors have also performed a negative control
test with isotype IgG-Alexa647 antibody to demonstrate a nonspecific
binding with PD-L1 protein. The individual photon burst signal from a
fluorescence dye was identified and counted as the analytical signal. The
concentrations of PD-L1 determined in channels of the microfluidic
biosensor, were 30.5 pM and 2.33 pM, for HeLa and MDA-MB-231 cells
lysates, respectively. From these values, the original PD-L1 concentra­
tions in single cells can be determined, on the bases of the numbers of
cells being analyzed (Ncells = 5×104), and single cell volumes of 4.7 pL
and 5.7 pL, for HeLa and MDA-MB-231 cells, respectively. Considering
the necessary dilutions of the cell lysates to prevent microchannel
clogging (9x and 5x dilutions, respectively), the cellular PD-L1 con­
centrations of 357 and 12.3 nM, for HeLa and MDA-MB-231 cells, were
obtained. Here, the cellular concentration of PD-L1 is defined as the total
number of moles of PD-L1 recovered during cell lysis from the cell
membrane and cytosol, divided by the single cell volume. The results of
measurements performed in tissues were consistent with those obtained
by conventional immunohistochemistry (IHC) staining. The concentra­
tions of PD-L1 in original tissues of patients under study yielded values
in the range from 0.73 to 5.35 nM for patient A and patient B, respec­
tively (Fig. 4). Thus, the proposed flow-proteometric microfluidic plat­
form offers a high sensitivity and a wide dynamic range. Other
advantages of the method include measurement simplicity, speed, and
reliability of protein determination in cells and tissues.
The detection of immune checkpoints, including PD-1, PD-L1, and
LAG-3, was also reported by Wuethrich et al., (2019). They have
developed a multiplexed immune checkpoint biosensor (MICB),
designed on the principle of a sandwich immunoassay with in situ
alternating-current electrohydrodynamic (ac-EHD) nanofluidic mixing
and sensitive colorimetric read-out, based on the enzymatic conversion
of 3,3′,5,5′-tetramethylbenzidine (TMB). The new class of low-cost and
stable protein capture agents, nanoyeast single chain variable fragments
(scFvs) specific to immune checkpoints PD-1, PD-L1, and LAG-3 were
Fig. 4. (A) Automated single-molecule-detection platform. (B) Immunohistochemistry (IHC) staining results and (C) PD-L1 concentration measurement by FAP
platform for PD-L1 expression in breast cancer tissue from patient A and patient B. Adopted from (Chou et al., 2018).
7
Biosensors and Bioelectronics 240 (2023) 115644
conjugated to the anti-hemagglutinin antibody functionalized electrode
surface. The sample was added together with horseradish peroxidase
(HRP)-conjugated secondary antibody against the immune checkpoint
proteins in PBS. The analytical color change signal was obtained after
the enzymatic oxidation of TMB in the presence of HRP. The TMB color
change from transparent to yellow (maximum absorbance wavelength
λmax = 450 nm) was observed after reaction quenching with sulfuric
acid. The limits of detection (LODs) for PD-1, PD-L1, and LAG-3 immune
checkpoint proteins, of 5 pg/mL, 5 pg/mL, and 50 pg/mL, were ach­
ieved, respectively. The proposed biosensor offers an advantage of the
parallel detection of several immune checkpoint markers and therefore,
is capable of detecting a multitude of biomarkers at once.
3.2. Optical systems for immune checkpoint biomarkers detection
A new optical biosensing system based on a sandwich-type SERS
platform was developed by Li et al., (2018). The Authors have utilized
the nanoyeast-scFvs (NYscFv) conjugated with streptavidin-coated
magnetic beads (MBs) for multiplexed detection of soluble cancer pro­
tein biomarkers including soluble programmed death 1 (sPD-1), soluble
programmed death-ligand 1 (sPD-L1) and soluble epidermal growth
factor receptor (sEGFR). The SERS tags consisted of the anisotropic gold
(Au)-silver (Ag) (Au-Ag) alloy nanoboxes (NBs) used for the signal
enhancement, Raman reporters: 4-mercaptobenzoic acid (MBA), 5,
5-dithiobis(2-nitrobenzoic acid) (DTNB), 2,3,5,6-tetrafluoro-4-mercap­
tobenzoic acid (TFMBA), which generate unique Raman signals at
1585 cm-1, 1335 cm-1 and 1376 cm-1, respectively, and anti-PD-1,
anti-PD-L1 and anti-EGFR polyclonal antibodies, used as the affinity
reagents (Fig. 5). The biosensor developed has enabled detection of
sPD-1, sPD-L1, and sEGFR, with the limits of detection (LOD) of 6.17
pg/mL, 0.68 pg/mL, and 69.86 pg/mL, respectively. The tests in spiked
human serum samples have also been performed with recovery rates
between 82.99% and 101.67%. Due to the extremely sensitive detection
of cancer biomarkers, this new SERS platform may be able to help in
early and timely diagnosing and cancer therapy.
Very sensitive surface plasmon resonance (SPR) transduction based
K. Ratajczak et al.
Fig. 5. Schematic illustration for the detection of sPD-1, sPD-L1 and sEGFR using SERS platform. Adopted from (Li et al., 2018).
biosensor for the detection of soluble PD-L1 (sPD-L1) immune check­
point biomarker has been designed by Luo et al., (2019). The
anti-sPD-L1 monoclonal antibodies (anti-sPD-L1 MAbs) were linked to
the surface of the sensor by the staphylococcal protein A (SPA). The SPR
immunosensor built on an excessively tilted fiber grating (ExTFG),
coated with large-sized gold nano-shells (~160 nm) capped with
cysteamine, was able to detect sPD-L1 down to an extremely low
detection limit, LOD = 1 pg/mL (0.04 pM) and LOD = 5 pg/mL, in
phosphate-buffered solution (PBS) and fetal bovine serum (FBS) sam­
ples, respectively. Although, the immunosensor was highly specific to
sPD-L1 molecules, the detection range was not satisfactory. The sensi­
tivity of the biosensor could be further improved by using graphene
oxide (GO) to encapsulate the gold nano-shells before immobilization on
the fiber grating. The reduction of the core/cladding diameter of ExTFGs
could also be applied for improving the sensitivity of the proposed
ExTFG-LSPR sensors. This genosensor’s advantages are also associated
with the determination speed and label-free detection of sPD-L1 anti­
gens, thus offering a potential application in cancer detection.
Aptamers against transmembrane protein PD-L1 have also been
synthesized to enable controlling the immune checkpoints. Yazdian-­
Robati et al., (2017) have developed new DNA aptamers (Apt5 and
Apt33) against the transmembrane protein PD-L1 using Protein-SELEX
method based on the Ni2+ complex with nitrilotriacetic acid (Ni-NTA)
adsorbed on agarose magnetic beads. The aptamer with better binding
affinity to the target (Apt5) was further investigated using flow cytom­
etry. The synthesized aptamer, labeled with ATTO 647 N red-emitting
fluorescence dye, was applied for the detection of A2780 human
ovarian carcinoma cell line. This fluorescent aptasensor enabled tumor
cells detection with LOD 10 cells/mL and linearity in the range from 50
cells/mL to 1000 cells/mL, which is comparable to the results of other
analytical methods for PD-L1 positive cancer cells determination, such
as the dual-aptamer target binding strategy, localized surface plasmon
resonance (LSPR), fluorescence, and electrochemical methods. There­
fore, the proposed method has a high potential for use in early stages of
cancer development. Furthermore, the aptamers are promising alter­
natives to antibodies due to the easy synthesis and such features as the
unique tertiary structure with high specificity and affinity towards
target molecules, no immunogenicity, no toxicity, resistance to the high
temperature, long storage time, as well as the ease of modification.
3.3. Plasmonic systems for immune checkpoint biomarkers detection
Recently, for the highly sensitive determination of proteins, a bio­
sensing technology, based on the modulation of resonance energy
transfer (RET), was proposed (Grel et al., 2020; Stobiecka, 2014; Sto­
biecka and Chalupa, 2015). The method was based on the modulation of
RET from a donor fluorescent dye molecule to a plasmonic gold nano­
particle (AuNP), acting as the acceptor, through a sub-monolayer
adsorption film formed by PD-L1 protein on a AuNPs. Voids in the
protein film acted as the gates for RET and enabled detection of PD-L1
8
Biosensors and Bioelectronics 240 (2023) 115644
down to 1.2 nM (Grel et al., 2020) (Fig. 6). The adsorption of PD-L1
on AuNP was observed due to the high affinity of PD-L1 to the surface
coating on the AuNP. It has been found that the high affinity of PD-L1 to
AuNP@Cit is associated with: (i) supramolecular interactions of amino
acids of the protein with citrates in AuNP coating, due to hydrogen
bonding, (ii) partial replacement of citrates from AuNP surface and
direct bonding of PD-L1 with Au surface atoms via Au-S covalent
bonding, due to some of the six cysteine residues present in the structure
of PD-L1; (iii) cation-mediated bridge formation, and (iv) strong elec­
trostatic screening of negative charges of the components by relatively
high ionic strength of the 50 mM buffer solution used.
The gRET method has the advantages of simple detection mechanism
and convenient operation while obtaining a very sensitive detection
limit in the buffer solution in absence of interferent proteins. However,
in biological matrix samples such as a serum, the implementation of
antibodies against PD-L1 attached to the gold nanoparticles have to be
applied for improvement of the method specificity toward the detection
of biomarkers in the body fluids. Also, for the simultaneous detection of
immune checkpoint proteins, different specific antibodies should be
immobilized onto gold nanoparticles surface.
4. Inhibitors for PD-1/PD-L1 immune checkpoint blockade
The binding properties of monoclonal antibodies (mAbs) against PD-
1/PD-L1, which are the key factors in the immune checkpoint inhibition,
can conveniently be investigated using the surface plasmon resonance
(SPR) technique. Such investigations have been performed by several
groups (Brown et al., 2020; Ghiotto et al., 2010; Luan et al., 2016; Tan
et al., 2018)
4.1. Antibodies as immune checkpoint inhibitor
Recently, Brown et al. (Brown et al., (2020) have investigated the
effect of chip type (flat or 3D-hydrogel) on the SPR-derived binding rate
constants and affinities of monoclonal antibodies (mAbs) against pro­
grammed cell death protein 1 (PD-1) to their specific target antigens.
The capture surface of chip was prepared by amine-coupling of anti­
bodies onto planar surfaces such as carboxymethyldextran exhibiting
the thickness < 5 nm (CMD-P), as well as a nearly flat surface such as
polycarboxylate hydrogel exhibiting the coating thickness about 30 nm
(HC-30M) or 3D-hydrogel such as carboxymethyldextran hydrogel,
exhibiting the coating thickness about 200 nm (CMD-200M). The Au­
thors have utilized in their investigations a lower throughput Biacore 8K
SPR instrument and a high-throughput Carterra LSA platform. A variety
of epitope coverage and ability of mAbs to block the direct interaction
between PD-1 and PD-L1 proteins was also studied. The Authors have
screened over thirty mAbs including camrelizumab (camre), cemiplimab
(cemip), pembrolizumab (pembro), nivolumab (nivo), and sintilimab
(sinti). They have shown, that only three of the antibodies (mAb05,
mAb12, and mAb30) were unable to block the binding to PD-L1 (Fig. 7).
K. Ratajczak et al.
Fig. 7. Network plots depicting the epitope clusters deduced from binning a panel of 31 anti-PD-1 mAbs. (A) The coloured bins represented by a family of mAbs
sharing an identical blocking profile, (B) PD-L1 blockade by mAbs (green = blocks, red = does not block). Adopted from (Brown et al., 2020), Creative Commons
Attribution License.
These label-free investigations enable to gain deeper insights into the
mechanism of action of new drugs under development for cancer
treatment.
4.2. Small molecules and peptides as immune checkpoint inhibitors
Due to the increasing number of cancer cases, the exploration of new
methods for tumor treatment is of great interest. Recently, in addition to
the mAbs utilized as the PD-1 and PD-L1 inhibitors, researchers have
also developed new peptides, organic molecules, and natural products
against the immune checkpoints. Such studies have been performed by
Liu et al., (2016). The Authors have discovered new small molecule
inhibitors of PD-1 by applying a homogeneous time-resolved fluores­
cence (HTRF) technique for investigation of the inhibitive activity of the
sulfamethizole and sulfamethoxypyridazine synthetic compounds. They
have employed the N,N-dimethylcarbamate and other alkyl-substituted
amines and resorcinol (a dihydroxyl phenol at meta-position) as the
9
Biosensors and Bioelectronics 240 (2023) 115644
Fig. 6. (A) Principle of gated resonance energy
transfer (gRET) (drawn not to scale); (B) Dependence
of the gRET efficiency for energy transfer from FITC
to AuNP@Cit on PD-L1 concentration showing satu­
ration at higher PD-L1 concentrations; (C) Details of
the limit of detection (LOD) determination using 3σ
method (LOD = 1.2 nM); CPD-L1 [nM]: (1) 0, (2) 1.27,
(3) 3.17, (4) 6.3, (5) 12.7, (6) 25.3, (7) 38, (8) 51, (9)
63.0; CFITC = 66.7 nM; CAuNP@Cit = 2.02 nM; λex =
495 nm. Adopted from (Grel et al., 2020), Creative
Commons Attribution (CC BY) license (http://cre
ativecommons.org/licenses/by/4.0/).
important core for developing the PD-1 inhibitors. These investigations
indicate that instead of monoclonal antibodies, other organic molecules
may be used and applied as the programmed cell death-1inhibitors in
cancer therapy. It opens a new way for the design and synthesis of a new
class of drugs.
Han et al., (2018) have used a SPR technique for the investigation of
the affinity and competitive inhibition of nine caffeoylquinic acid
compounds (CQAs) against PD-1/PD-L1. They have found four small
molecules, including 1-CQA, 3-CQA, 4-CQA, and 5-CQA, that could act
as the PD-1/PD-L1 inhibitors. The 3-CQA, 4-CQA and 5-CQA inhibitors
revealed micromolar inhibition with low IC50 values equal 36.6, 38.3,
and 45.6 μM, respectively, and 1-CQA has shown inhibition of
PD-1/PD-L1 with somewhat higher IC50 value of 87.3 μM (Fig. 8). Thus,
SPR technology, due to its outstanding properties, such as the speed,
sensitivity, and label-free measurements, has been explored for effective
and rapid screening of small molecules to find the immune checkpoint
inhibitors and, thereby, SPR has become the method utilized in cancer
K. Ratajczak et al.
Fig. 8. (A-B) The example of competitive inhibition of two CQAs (1-CDQA (A) and 4-CDQA (B) on PD-1/PD-L1. CPD-1 = 392 nM; (C) Structures of 1-CQA, 3-CQA, 4-
CQA, 5-CQA; (D) The IC50 curves of small molecules including 1-CQA, 3-CQA, 4-CQA, 5-CQA on PD-1/PD-L1. Ferulic acid is a negative control. Adopted from (Han
et al., 2018).
treatment.
A phage display biopanning strategy for screening of small peptide-
based anti-PD-L1 inhibitors (CLP001, CLP002, CLP003, CLP004) has
been proposed by Cheng’s group (Liu et al., 2019). This method relies on
the expression of peptide or protein sequence by each phage in phage
library. The strategy developed is based on binding of the peptide under
study to specific residues on a target protein (PD-L1), and subsequently
blocking the protein’s interaction with its PD-1 receptor. The binding
affinities of peptides for human PD-L1 protein were determined using
surface plasmon resonance technique. The system was based on the
PD-L1 protein covalently immobilized by amine coupling method with
dextran hydrogel functionalized with COOH groups onto a gold chip.
The equilibrium dissociation constants (KD), determined for CLP001,
CLP002, CLP003 and CLP004 peptides bound to a human PD-L1 protein
were: 534, 366, 117, and 544 nM, respectively. The antitumor activity of
peptides was evaluated using the CT26 colorectal tumor-bearing mice
and the tumor penetration capability in MDA-MB-231 cells by
anti-PD-L1 peptides was also investigated. The phage display procedure
was successfully applied for the discovery of small peptide-based
checkpoint inhibitors; thus, it has a potential for application in cancer
therapy. The developed CLP002 peptide has been found to bind specif­
ically to PD-L1 protein and due to the PD-1/PD-L1 interaction blocking,
has exhibited a better tumor penetration in a 3D tumor spheroid model
in comparison with the anti-PD-L1 antibody. Furthermore, it inhibited
the tumor growth and increased the survival of CT26 mice model.
5. Nanoparticle carriers for drug delivery technologies to
suppress PD-1/PD-L1 activity
The expression of PD-L1 protein on the surface of a variety of cancer
cells causes the inhibition of kinase signaling pathways upon binding of
these ligands to the immune cell PD-1 receptors and results in sup­
pressing of the T cells activity and taming of immune response. In this
way, cancer cells expressing PD-L1 can easily evade immune reaction of
10
Biosensors and Bioelectronics 240 (2023) 115644
the host and grow freely despite the presence of T cells (Grel et al., 2020;
Santarpia and Karachaliou, 2015). Therefore, developing brakes capable
of targeting the PD-1/PD-L1 pathway is highly desired and has become
the most extensively researched strategy for cancer treatment. Also, the
new technologies for immune checkpoint brakes delivery are urgently
needed. In addition to the anti-PD-L1 antibodies used as the brakes for
the direct blocking of PD-L1 protein, an alternative therapy based on
nanodelivery of small interfering RNAs (siRNAs) to cancer cells has
recently been applied. For a more effective targeted delivery system and
less toxic cancer treatments, the functionalized nanoparticles have been
used (Kosmides et al., 2017; Kwak et al. 2017, 2019; Li et al., 2019b). In
Table 3 the most important features of nanocarriers and guiding prin­
ciples for their improvement/creation are listed.
A dual immunoswitch, based on an 80 nm iron-dextran particle
platform coated with anti-PD-L1 and anti-4-1BB antibodies, has been
developed by the Schneck’s group (Kosmides et al., 2017). These
nanoparticles were able to switch off the immunosuppressive PD-L1
pathway on tumor cells and switch on the 4-1BB pathway on CD8+ T
cells causing inhibition of tumor growth and are more effective than
intratumoral injection of soluble antibodies. These immunoswitch
nanoparticles enhance the efficacy of two immunotherapies and are thus
more effective at low doses in cancer therapy.
The simultaneous silencing of PD-1 and PD- L1 expressions on
cytotoxic T cells (CTLs) and colon tissues, respectively, have been pro­
posed by Kwak et al., (2019). The Authors have used the poly
(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with siRNAs
against PD-1 and its ligand PD-L1 gene (PD-1 siRNA/PD-L1 siR­
NA@PLGA NPs) for codelivery of PD-1 and PD-L1 siRNAs. The Authors
have found that the co-inhibition of PD-1 and PD-L1 results in enhanced
antitumor effects in comparison with a single silencing of PD-1 or PD-L1
alone, indicating that the concerted inhibition is preferable (Fig. 9). The
apoptosis process was monitored by the TUNEL staining assay.
A nanocarrier system for co-delivery of PD-L1 siRNA (siPD-L1) and
an 1-methyl-DL-tryptophan (1 MT) indoleamine 2,3-dioxygenase (IDO)
K. Ratajczak et al. Biosensors and Bioelectronics 240 (2023) 115644

Table 3
Advantages and guiding principles of nanocarriers to suppress PD-1/PD-L1 activity.
Nanoparticles Advantages Guiding principles for improvement/creation of Ref.
nanoparticles/disadvantages

stem cell membrane (SCM) camouflaged
polydopamine (PDA) nanoparticles carrying
doxorubicin (DOX) and PD-L1 siRNA -
PDA–DOX/siPD-L1@SCM NPs -
80 nm iron-dextran nanoparticles with the
antibodies against 4-1BB (a co-stimulatory
receptor found on the effector T cells) and
antibodies against PD-L1 (found on the cancer
cells)
Poly (lactic-co-glycolic acid) (PLGA) nanoparticles
loaded with PD-1 and PD-L1 siRNA
siPD-L1 with a polymeric carrier (dubbed “pd”)
consisting of disulfide-cross-linked polyethy-
leneimine (CLPEI) and dermatan sulfate (DS) -
siPD-L1/pd
- efficient loading of DOX and PD-L1 siRNA
via π–π stacking interactions,
- reduction of the DOX toxicity,
- synergistic therapeutic effect,
- good compatibility with blood,
- simultaneously block the inhibitory
checkpoint PD-L1 signal and stimulate T cells
via the 4-1BB co-stimulatory pathway,
- significantly delay tumor growth,
- extend survival in multiple in vivo models of
murine melanoma and colon cancer,
- increased density, specificity, and in vivo
functionality of tumor-infiltrating CD8+ T
cells checkpoint blockade efficacy,
- synergy between the two immunotherapies,
- effective at low doses,
- target two different cell types in the tumor
microenvironment,
- significant tumor growth suppression and
long-term tumor inhibition in colon cancer
by co-silencing of PD-1 and PD-L1,
- co-inhibition of PD-1 and PD-L1 exhibited
the enhanced antitumor effects,
- suppression of the expression of PD-L1,
- attenuation the expression of tumor-specific
genes in B16F10 cells,
- suppression of melanoma growth in immune-
compromised nude mice,
- good hydrophilicity, biocompa-tibility, stability and (Mu et al.,
biodegrade-bility of dopamine and spontane-ously 2021)
polymerization to polydo-pamine (PDA) nanoparticles
(NPs),
- large number of catechol and amino functional groups in
PDA for binding many functional molecules to the
surface,
- loss of DOX during the physical extrusion process,
- combinatorial immunotherapy for cancer treatment as (Kosmides
promising success in clinical trials, et al., 2017)
- use of nonspecific immunomodulators requires high
doses of the drugs and results in significant off-target side
effects,
- low target specificities, very long half-lives, risk of an (Kwak et al.,
autoimmune response, and limitations of the antibody 2019)
manufacturing process,
- less likely to respond to single checkpoint blockade,
- specific toxicity of the CLPEI/DS (dubbed “pd”) to a (Kwak et al.,
group of cancer cells that express CD146, 2017)
- high target specificity of siRNA,

inhibitor for PD-1/PD-L1 blockade has been developed by Li et al.,
(2019b). The tumor-targeted system was based on micelles with func­
tional penetrating peptides (Lin TT1, sequence: AKRGARSTA) conju­
gated with cholesterol and histidine 7 (Chol-HHHHHHH-AKRGARSTA,
CHL). The antitumor effect of nanoparticles was evaluated in vitro and in
vivo.
The suppression of PD-L1 expression in melanoma cells has also been
achieved by delivering siPD-L1 with a polymeric nanocarriers consisting
of disulfide-crosslinked polyethyleneimine (CLPEI) and dermatan sul­
fate (DS) (siPD-L1/pd) by Kwak et al., (2017). This nanodrug was
applied to the melanoma growth in immune-compromised mice and the
interferon-γ (IFN-γ)-challenged B16F10 melanoma cells. A successful
suppression of PD-L1 expression in B16F10 melanoma growth in
C57BL/6 immune-competent mice was also reported.
6. Exosomal PD-L1 as a novel therapeutic approach in cancer
treatment
Exosomes are extracellular vesicles (30-100 nm in diameter) that are
released from cells and contain a rich variety of bioactive molecules
dependent on the type and state of the cell of origin. Recently, several
research groups have discovered that the PD-L1 is also expressed on the
surface of exosomes released from different kinds of cancer cells (Chen
et al., 2018; Kim et al., 2019; Poggio et al., 2019; Song et al., 2019;
Stobiecka, 2015; Zhang et al., 2023b). Flow cytometry and immuno­
fluorescence results suggested that PD-L1 is present not only on the
surface of vesicles but also within the vesicle-like structures (Xie et al.,
2019; Poggio et al., 2019). The level of PD-L1, carried on the exosome
surface, was also associated with tumor progression in a variety of
cancers including melanoma (Gyukity-Sebestyén et al., 2019), breast
cancer (Xie et al., 2019), and head and neck squamous cell carcinoma
(HNSCC) (Theodoraki et al., 2018). It was also shown that exosomal
PD-L1 secreted from cancer cells has immunosuppressive effects (Tang
et al., 2020; Morrissey and Yan, 2020). Poggio et al., (2019) have
observed suppression of T cell activation in a draining lymph node and
PD-L1-deficient tumor cells. Chen et al., (2018) have shown that the
exosomal PD-L1 suppresses anti-tumour immunity systemically and the
circulating exosomal PD-L1 could become a predictor for the clinical
outcomes of anti-PD-1 therapy. Moreover, the exosomal PD-L1 can be
considered as a negative prognostic factor for pancreatic ductal adeno­
carcinoma (PDAC) patients (Lux et al., 2019). Because of the exosomes
significant cargo, the tumor-derived exosomes could potentially be used
to form a pool of tumor antigens to stimulate the anti-tumor response
(Xie et al., 2019).
Therefore, targeting of the exosomal PD-L1 expression can become a
novel therapeutic approach in future cancer treatment. Li et al., (2019a)
have demonstrated that the level of exosomal PD-L1 in non-small cell
lung cancer (NSCLC) patients was higher than in healthy controls and
correlated positively with tumor size, positive lymph node status, distant
metastasis and advanced TNM stage (Fig. 10). Exosomes were isolated
according to manufacturer protocol from serum collected from blood
samples using the commercial kit (Total Exosome Isolation Kit) by
centrifugation.
6.1. Detection of exosomal PD-L1 using optical biosensors
Recent endeavor to elucidate the mechanisms of cancer metastasis
has led to the discovery of exosomal intercellular communication (Sto­
biecka, 2015; Chung et al., 2020) and a wealth of cancer biomarkers (Li
et al., 2020) carried by exosomes secreted by cancer cells. Hence, the
detection of exosomal proteins becomes a new means of non-invasive
cancer screening, staging, and monitoring. The detection of exosomal
proteins, such as the exosomal epidermal growth factor receptor (EGFR)
and PD-L1, using the intensity-modulated compact SPR biosensors, was
reported by Liu and colleagues (Liu et al., 2018). The exosomes were
isolated from human serum samples using the exosome kit and the ob­
tained exosome pellets were then resuspended in PBS buffer. The SPR
biosensor was based on a gold biochip modified with a mixture of

11
K. Ratajczak et al.
Fig. 9. (A) Simultaneous silencing of PD-1 and PD-L1 by systemic delivery of siRNA for suppression of colon tumor growth; (B) In vivo antitumor effects of siR­
NA@PLGA NPs. TUNEL staining assay demonstrating apoptotic cells in the MC38 tumor sections from the injected mouse groups. Scale bar = 50 μm. Adopted from
(Kwak et al., 2019).
methyl-polyethylene glycol-thiol, biotinylated-polyethylene glyco­
l-thiol, NeutrAvidin, and antibodies, including biotinylated anti-EGFR,
biotinylated anti-PD-L1, and biotinylated anti-IgG. The exosomal pro­
teins were detected by incubating exosomes on the biochip surface. The
Authors have demonstrated better detection sensitivity of compact SPR
biosensor than ELISA, while providing similar sensing accuracy. They
have shown that ELISA was not able to detect exosomal PD-L1 levels in
serum samples. The EGFR and PD-L1 biomarkers for cancer diagnostics
were detected in cell culture medium and serum samples from
non-small-cell lung cancer (NSCLC) patients and normal controls
(Fig. 11).
A simple analytical method for determination of exosomal PD-L1
directly from human serum samples was proposed by Pang et al.,
(2020). The system was based on Fe3O4@TiO2 nanoparticles and
anti-PD-L1 antibody-modified Au@Ag@MBA nanoparticle SERS tags.
The method allows to detect a single programmed death ligand-1 posi­
tive (PD-L1+) exosome/μL within 40 min. The method also enabled to
distinguish the early and late nonsmall cell lung cancer (NSCLC) patients
from healthy controls. Human serum samples were filtrated with a 0.22
μm syringe-driven filter, incubated with the nanoparticles for exosome
isolation, and labeled with SERS tags.
6.2. Detection of exosomal PD-L1 using electrochemical biosensors
Cao et al., (2020) have detected PD-L1+ exosomes with a low limit of
detection 334 particles/mL using porous nanomaterials consisted of
horseradish peroxidase (HRP) encapsulated into zeolitic imidazolate
framework-8 (ZIF-8) coating by polyvinylpyrrolidone (PVP),
PVP@HRP@ZIF-8. The Authors have attained the enhancement of the
12
Biosensors and Bioelectronics 240 (2023) 115644
signal, by utilization of an efficient DNA amplification based on in situ
hyperbranched rolling circle amplification (HRCA). During the HRCA
process, the hydrogen ions are sustainably released along with the
continuous addition of nucleotides caused the pH of the medium
lowering and disassembly of PVP@HRP@ZIF-8 at a mildly acidic envi­
ronment, followed by the release of HRP. A high electrochemical
response was observed in the presence of PD-L1+ exosomes, during
HRP-catalyzed oxidation of o-phenylenediamine (OPD) with the addi­
tion of hydrogen peroxide (H2O2). The proposed method was also
enabled to detect the elevated levels of circulating PD-L1+ exosomes in
undiluted serum samples from healthy controls, nonmetastatic breast
cancer patients and metastatic breast cancer patients.
6.3. Exosomes as a drug delivery carriers
Recently it was also shown that the exosomes due to their low
toxicity, high bioactivity, an innate stability, low immunogenicity and
excellent tissue/cell penetration capacity as well as biocompatibility
become a promising drug delivery carriers (Zhang et al., 2023a; Lee
et al., 2022; Sen et al., 2023; Chen et al., 2021; Kar et al., 2023).
Mu et al., (2021) have developed mesenchymal stem cell membrane
(SCM)-derived vesicles coated polydopamine (PDA) nanoparticles (NPs)
as an efficient tumor-targeting delivery platform. The nanoparticles
have encapsulated doxorubicin (DOX) chemotherapeutic drug and
siRNA targeting PD-L1 (siPD-L1) loaded onto the surface of PDA NPs via
π–π stacking interactions (PDA-DOX/siPD-L1@SCM NPs). The Authors
have shown that the PD-L1 protein on the surface of PC-3 cells was
effectively suppressed by codelivered PD-L1 siRNA and the treatment of
mice with PDA–DOX/siPD-L1@SCM effectively inhibit the growth of
K. Ratajczak et al. Biosensors and Bioelectronics 240 (2023) 115644

Fig. 10. (A) TEM image of exosomes (black arrows)

isolated from NSCLC patients, (B) Schematic diagram
of the formation and secretion of exosomes with PD-
L1 by tumor cells, (C-D) Quantitative analysis of
exosomal PD-L1 (Exo-PD-L1) levels (pg/mL serum)
(C) and relative Exo-PD-L1 levels (pg/mg exosomal
protein) (D), among healthy individuals (n = 27),
stage I–II (n = 57) and III/IV (n = 28) NSCLC pa­
tients; *p<0.05; ***p<0.001; #p<0.05; ##p<0.01.
Adopted from (Li et al., 2019a), Creative Commons
Attribution 4.0 International License (http://creati
vecommons. org/licenses/by/4.0/).

Fig. 11. (A) Setup of compact SPR biosensor (left)

and the photo and schematic diagram of the biochip
(right); (B) Sensing mechanism of compact SPR
biosensor; (C) Representative real-time response
curve of compact SPR biosensor detecting exosomal
PD-L1 in serum sample from a Stage III lung cancer
patient; (D) Expression of exosomal PD-L1 in serum
samples measured by compact SPR biosensor. 50 μL
serum sample was used in all measurements. n = 5, *:
p < 0.05). Adopted from (Liu et al., 2018).

13
K. Ratajczak et al.
prostate cancer (PCa) bone metastases in nude mice.
7. Conclusions and future outlook of immune checkpoint
blockade in cancer treatment
The remarkable immune checkpoint inhibitors, developed recently
to control the organism’s own immune responses, have become the key
elements of the emerging methods of cancer immunotherapy. In parallel
with these achievements, extensive works have been carried out to
design and test novel biosensors to monitor and screen checkpoint in­
hibitors, and to detect a range of cancer biomarkers to enable quick
evaluation of the cancer treatment. However, there are many factors
implicated in the immune checkpoint inhibitors response including
circulating immune factors (diverse white blood cells (WBCs)),
emerging germline genetic traits, (human leukocyte antigen class I
(HLA-I) complex), phenotypic host factors (obesity, cholesterol, gender),
environmental factors (medications), human gut microbiota (Gunjur
et al., 2022). The blocking of immune checkpoints by monoclonal an­
tibodies has been particularly effective in improving clinical outcomes
for patients with some types of cancers but there is still a long way to
attain the expected high efficacy of immunotherapy for most cancers. In
view of the assessment of the progress made in the development of
PD-1/PD-L1 pathway-based anticancer immunotherapeutic methods,
performed in this work, it is evident that mAbs against PD-1/PD-L1
demonstrate a great efficacy in cancer treatment in case of cancers,
such as melanoma, hepatocellular carcinoma, Hodgkin’s lymphoma,
and NSCLC. We envision that further development of the analytical
techniques for fast, convenient, and highly sensitive monitoring of im­
mune checkpoint inhibitors, such as the gated-RET, SERS, optical and
microfluidic techniques, will enable precision control over the cancer
treatment. Another method of controlling the immune response involves
downregulation of tumor PD-L1 expression by gene-silencing utilizing
siRNA. Since a pronounced expression of PD-L1 has been found on the
surface of exosomes, which were released from cancer cells, the exoso­
mal PD-L1 level now turns out to be now a diagnosis and predictive
biomarker for cancer patients’ survival rate in a noninvasive “exosomal
biopsy”. Although not yet applied in clinical practice, the exosomal bi­
opsy is expected to become soon one of the most important, unique, and
robust routines in diagnostics, staging, and providing reference infor­
mation for real-time monitoring and progression of cancers. There are
now some commercially available products/kits with their own specific
protocols for the isolation of exosomes from bodily fluids. They can be
utilized for the detection and analysis of genetic or proteomic bio­
markers (Mohammadi et al., 2021; Ohannesian et al., 2020; Lai et al.,
2022). Furthermore, due to a high drug-carrying capacity, non-cytotoxic
effects, and a low immunogenic profile of exosomes, we believe that the
encapsulation of anticancer drugs and immune checkpoint inhibitors in
exosomal nanocarriers for targeted drug delivery is likely to be one of
the most exciting paths leading in the near future to novel cancer
therapies aiming at prevention of metastasis which is the key point for
improving the patients’ survivability.
Declaration of competing interest
The authors declare no conflict of interest.
Data availability
No data was used for the research described in the article.
Acknowledgments
This research was supported by funding provided by the Program
OPUS of the National Science Centre, Poland, Grant No. 2017/25/B/
ST4/01362. Financial support from the Foundation for Polish Science
(FNP) through START program (to K.R.) and Program SONATA BIS of
14
Biosensors and Bioelectronics 240 (2023) 115644
the National Science Centre, Poland, Grant No. 2019/34/E/ST4/00281
(to S.J.) is also acknowledged.
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