biosensors

Review
Nanotechnology-Enabled Biosensors: A Review of Fundamentals,
Design Principles, Materials, and Applications
Manickam Ramesh 1, * , Ravichandran Janani 2 , Chinnaiyan Deepa 3 and Lakshminarasimhan Rajeshkumar 4

1 Department of Mechanical Engineering, KIT-Kalaignarkarunanidhi Institute of Technology,

Coimbatore 641402, Tamil Nadu, India
2 Department of Physics, KIT-Kalaignarkarunanidhi Institute of Technology,
Coimbatore 641402, Tamil Nadu, India
3 Department of Artificial Intelligence & Data Science, KIT-Kalaignarkarunanidhi Institute of Technology,
Coimbatore 641402, Tamil Nadu, India
4 Department of Mechanical Engineering, KPR Institute of Engineering and Technology,
Coimbatore 641407, Tamil Nadu, India
* Correspondence: mramesh97@gmail.com

Abstract: Biosensors are modern engineering tools that can be widely used for various technological
applications. In the recent past, biosensors have been widely used in a broad application spectrum
including industrial process control, the military, environmental monitoring, health care, microbiology,
and food quality control. Biosensors are also used specifically for monitoring environmental pollution,
detecting toxic elements’ presence, the presence of bio-hazardous viruses or bacteria in organic matter,
and biomolecule detection in clinical diagnostics. Moreover, deep medical applications such as
well-being monitoring, chronic disease treatment, and in vitro medical examination studies such
as the screening of infectious diseases for early detection. The scope for expanding the use of
biosensors is very high owing to their inherent advantages such as ease of use, scalability, and
simple manufacturing process. Biosensor technology is more prevalent as a large-scale, low cost, and
enhanced technology in the modern medical field. Integration of nanotechnology with biosensors
has shown the development path for the novel sensing mechanisms and biosensors as they enhance
the performance and sensing ability of the currently used biosensors. Nanoscale dimensional
integration promotes the formulation of biosensors with simple and rapid detection of molecules
Citation: Ramesh, M.; Janani, R.; along with the detection of single biomolecules where they can also be evaluated and analyzed
Deepa, C.; Rajeshkumar, L. critically. Nanomaterials are used for the manufacturing of nano-biosensors and the nanomaterials
Nanotechnology-Enabled Biosensors: commonly used include nanoparticles, nanowires, carbon nanotubes (CNTs), nanorods, and quantum
A Review of Fundamentals, Design dots (QDs). Nanomaterials possess various advantages such as color tunability, high detection
Principles, Materials, and sensitivity, a large surface area, high carrier capacity, high stability, and high thermal and electrical
Applications. Biosensors 2023, 13, 40. conductivity. The current review focuses on nanotechnology-enabled biosensors, their fundamentals,
https://doi.org/10.3390/ and architectural design. The review also expands the view on the materials used for fabricating
bios13010040
biosensors and the probable applications of nanotechnology-enabled biosensors.
Received: 29 November 2022
Revised: 22 December 2022 Keywords: biosensors; nanotechnology; nanomaterials; carbon nanotubes; quantum dots; biosensing
Accepted: 23 December 2022
Published: 27 December 2022

1. Introduction
Copyright: © 2022 by the authors.
Biosensing is a traditional concept that is inherently present in many life forms, and
Licensee MDPI, Basel, Switzerland. it is used for protection against predators and harsh environments when seen through
This article is an open access article an evolutional viewpoint. Some examples of inherent biosensing in organic life forms
distributed under the terms and include toxic sensibility in certain algal species, the electro-sensitive nature of sharks,
conditions of the Creative Commons and the inherent augmented offensive capability of canines [1–3]. As per the definition
Attribution (CC BY) license (https:// coined by the International Union of Pure and Applied Chemistry (IUPAC), a biosensor
creativecommons.org/licenses/by/ is a device that uses a precise biochemical reaction arbitrated by the immune system,
4.0/). isolated enzymes, organelles, or tissues for the detection of chemical compounds through

Biosensors 2023, 13, 40. https://doi.org/10.3390/bios13010040 https://www.mdpi.com/journal/biosensors

Biosensors 2023, 13, 40 2 of 32

the sensing of optical, thermal, or electrical signals [4]. Figure 1 shows the components of a
typical biosensor.

Figure 1. Biosensors and their components (reprinted from Ref. no. [1], copyright 2021, MDPI).

Various combinations of biomaterials and sensor elements are possible for manufac-
turing a biosensor for different sensing applications. Even though a broad spectrum of
materials can be used for the preparation of a biosensor, the fabrication process of a material
that goes in line with the requirements of a biosensor is very complex, and this limits the
manufacture of biosensors [5–7]. Hence, the study of compatible materials for biosensing
aligned with biosensors’ requirements and the investigation of ease of fabrication is highly
required. This case is more prevalent in liquid crystal (LC)-based biosensors as they are
purely material-dependent biosensors. The optical properties of LC biosensors are greatly
influenced by the reorientation of the aligned molecules which in turn depends on the
physical and chemical surroundings of the biosensor; LCs are more commonly used for
fabricating biosensors. It has been stated in various studies that LC-based biosensors find
their applications in sensing novel properties [8–10].
Researchers have found that the LC biosensors are very interesting lately owing to the
new avenues created by biosensors used for novel applications. As LC-based biosensors
depend purely on molecular arrangement, they have been in the limelight for the past
two decades, surpassing conventional biosensors for their utilization in a broad range of
applications owing to their enhanced sensitivity [11,12]. When the light is made to go
in a direction similar to the sample molecules in a polarizing optical microscope (POM)
under which the LC was placed in vertical alignment, it would be blocked by the polarizer
rendering a dark pattern. On contrary, if the analyte was kept along with the biosensor
and observed in the POM, a parallel or random angular arrangement in oblique alignment
was observed while the incident light falling over the LC material fragmented into two
polarized lights in a linear direction. Such optical anisotropy of the LC materials induced
rapid sensitivity and ease in visualization of the response in LC-based biosensors [13,14].
Electro-chemical biosensors are another class of biosensors that are typically used on
biological analytes. These biosensors react with the recognition elements of the biological
analytes and an electrical signal is produced for the transduced chemical response. These
types of biosensors find their major application in environmental, clinical, quarantine,
and pharmaceutical fields owing to various advantages such as miniature and portable
technology compatibility, high sensitivity, low cost, and faster response [15,16]. Many
of the commonly used commercial electro-chemical biosensors such as DNA hybridized
biosensors, immuno-sensors and enzyme-based biosensors use biological sensing materials
Biosensors 2023, 13, 40 3 of 32

which possess high selectivity and sensitivity rates. Despite various advantages, electro-
chemical biosensors have been built with some limitations including the high cost of
the biomolecules, being highly susceptible to different environmental conditions such as
oxygen content, devitalizers, pH, and temperature, lower long-term stability, complex
immobilization procedure, and instability towards certain chemicals. In order to dodge
the above disadvantages, electro-chemical biosensors have been built using elements with
nonbiological recognition, such as transition metal oxides (MXenes) which have been
used majorly during recent times owing to their outstanding stability, high selectivity, and
sensitivity [17–19]. The current review focuses on nanotechnology-based biosensors, the
materials used for the fabrication of nanobiosensors, and their potential applications. This
article elaborates on the challenges faced during the full-scale implementation of biosensors
and their prospects.

2. Nanotechnology—An Overview
Nanotechnological advances have paved the way for developing devices at the
nanoscale level using various nanomaterials which directly interact and are in contact
with the biomolecules or analytes for which the biosensors are intended to be used. Such
biosensors have many stand-alone properties including customized magnetic, electrical,
and optical properties, enhanced electrical conductivity, high sensitivity, and a low response
time when compared with the traditionally used biosensors. Hence, such biosensors can be
used in different bioengineering applications such as drug delivery applications [15,16].
Nanotechnology has been proven to be influential in medical fields such as disease detec-
tion with the aid of resonance, electrochemical, magnetic, electromechanical, thermal, and
optical methods [17]. It can also be stated that integration of nanotechnology in the field of
biosensing can offer various merits including a large surface-to-volume ratio, manifestation
of biological transduction and signaling mechanisms, and electro-chemical properties [18].
In recent times biosensors have witnessed huge advancements owing to the extensive
advents in transducers, nanotechnology, and signal amplifying techniques. Nevertheless,
biosensors are characterized by inherent irregular signal noises. A few biosensors are
highly dependent on aptamers or antibodies as bioreceptor molecules and due to this, their
shelf-life is affected resulting in poor sensing stability. Commercialization of biosensors
is also restricted by the reliability and accuracy of many of the modern biosensors [19,20].
Researchers are trying deeply to overcome this difficulty in many possible ways to enhance
the performance of biosensors. Various studies have focused on the machine learning (ML)
approach for analyzing sensing data. ML paves the way for the biosensors to abduct with
their currently prevailing challenges by turning the typical biosensor into an intelligent
biosensor and this works in such a way that it predicts the analyte concentration or species
based on a decision algorithm. In a few other studies, chemometrics was given as a prime
focus to analyze the response of a biosensor. Chemometrics is an extensively used chemical
analyzing technique that uses mathematical or statistical methods to analyze and render
maximum chemical data from the analysis of chemical information and to optimize and
select the experimental design to bring out the optimal process of measurement [2,21,22].
It has been stated in many studies that ML can effectively process big sensing data in
the form of complex samples or matrices. Some advantages of the ML method of chemical
analysis include the rendering of data from low-resolution and noisy sensing data that
may also have overlapped with one another. Besides these advantages, when ML methods
are deployed on a full scale, the hidden relations existing within the analyte parameters
and signals can be discovered with the aid of exploring the interrelations between the
bio-events and the signals and through various data visualization methods. ML methods
are usually used to interpret the raw form of sensing data obtained from the biosensor in
the following ways:
Biosensors 2023, 13, 40 4 of 32

• Categorization—Signals obtained from the sensing data are classified into different
types using various algorithms depending on the type of target analyte.
• Anomaly detection—The operating conditions of a biosensor and the sample ma-
trix significantly affect the functions of a biosensor and the contaminations interfere
with the sensing signal if the biosensor is deployed in the target site. In such cases,
the ML algorithm checks the correctness of the obtained signal, and if found incor-
rect, the signals which are interfered by the biofouling are corrected to improve the
sensor performance.
• Reduction of noise—Sensed signals are commonly embedded with noise. If the sensed
signal is interfered with by electrical noise, the signal changes in a few seconds or
minutes which makes it shift to a sub-second timeline. If ML models are trained to
detect noise, the sensing signal accuracy can be substantially improved.
• Pattern recognition and object identification—Then signals obtained by the biosensors
can be effectively and easily interpreted through the discovery of latent patterns and
objects with the aid of ML algorithms [23,24].

3. Fundamentals
3.1. Biosensors
To monitor biological reactions, biosensors have been used to analyze the interaction
and transform it into an electrical impulse. Electro-chemical, fiber optic, piezo, sound, and
thermal transducers along with biological components including enzymes, DNA, RNA,
metabolites, cells, and oligos are combined in biosensors [25,26]. Basically, a biosensor is an
analytical tool that detects and responds to changes in biological systems by producing an
electrical impulse. Enzymes, tissues, bacteria, cells, acids, and so on are all examples of bio-
logical processes. The voltage or current would be the transducer’s signal [27–29], based on
the kind of enzymes and the raw material utilized in the synthesis of organic compounds.
The major strength of biosensors is their capacity to transform biological interactions
into a type of electric signal that can be detected and quantified. It is also crucial to focus
on the effective analysis of minute variations throughout the biological processes when
different biomolecules combined [30,31]. Based on the numerous advantages, biosensors
are now being invented for their usage in the accurate diagnosis of infections and the
evaluation of food standards, as well as other applications in the environmental sector.
In medical field, biosensors have been developed to detect tumors, viruses, pollutants,
and biomarkers so as to diagnose diseases at an early stage [32,33]. However, biosensors
have become more significant because of their advantageous properties, such as cheap
production costs, quick reaction times, mobility, and the capability to measure biological
materials at a tiny scale with a high level of proficiency and sensitivities [34,35].
It has taken a long time for biosensor technology to advance for the study of other
physical components. To summarize, biosensors have numerous sections or components,
including the analyte, bioreceptor, transducer, electronics, and reader display. In most
biosensors, the reader display is connected to the electronics or signal processors which
are required to interpret the data [36,37]. As each biosensor operates on different working
principles, every reader must be conceived and built from scratch. Often, this is the costliest
aspect of developing sensors. As the sensor evaluates the consumer in response to different
stimuli, the transducer records the information and converts the stimulus into an electrical
impulse that can be measured as output data [38].
Biosensors find their usage in a wide variety of applications, including waste man-
agement, health monitoring, agricultural experimentation, forensics, biological testing,
and water quality control. Biosensory medical clothing is often used for overall health
observation, diseases diagnosis, and clinical evaluation [39,40]. Overwhelmingly, glucose
biosensors have been used to monitor and control diabetes. Blood sugar levels, a key
indicator of diabetes risk, are also monitored using biosensors. Biosensors’ continued
significance is shown by the fact that they enable patients to maintain their desired blood
sugar levels and enable researchers to trace the disease’s ecological impact. Biosensors
Biosensors 2023, 13, 40 5 of 32

may speed up disease diagnosis and patient monitoring [41,42], adding value to traditional
medical treatment.

3.2. Nanobiosensors
Nanoparticles are integrated during fabrication and the resulting biosensors are called
nano-biosensors. Nanomaterials are always the most investigated and examined of these
because of the wide range of bioanalytical activities they provide in fields such as bio-
imaging, diagnostics, medication administration, and the treatment that they enable [43,44].
Amperometric equipment has been used to assess enzyme-based reactions, whereas fluo-
rescent QD devices are being used for measuring the binding efficacy and immunolabeling
applications that utilize conjugated nanoparticles to analyze biomolecular interactions. The
inherent optical features of nanoparticles as well as the potential ability to be coupled to
fluorescent markers make them a promising biosensor. Electro/chemiluminescent tests,
fluorescent-based tests, and biological field-effect transistors (bio-FET) tests can end up in
making CNTs, and other relevant carbon-based nanostructures including reduced graphene
oxide (rGO), graphene, and graphene sheets. Fluorescent-based assays are usually detected
through the quenching characteristics of graphene [45,46], and carbon nanomaterials can
adopt a variety of structures depending on the method of measurement being used.
Based on their size and form, which defines their fluorescence qualities, QDs provide
a wide range of applications in the areas of sensors and imaging. Functionalizing QDs with
polymers such as PEG and polysaccharides is required for specific identification. QDs have
several advantages over conventional dyes, including a higher yield of molar extinction and
quantum coefficient, a wider range of absorption and smaller spectra of emission, photo-
bleach resistance, and so on [47]. They are widely utilized in FET as well as in sandwich
assays. In addition to the nanostructures already described, there are several distinct
nano-based biosensors that may be used for a variety of purposes. Unusual applications
of nanotechnology for biosensing seems to be the development of glucose sensors for
people with diabetes, the identification of antigens of HIV/AIDS, the monitoring of the
bioburden of microbes in cases of infection in the urinary tract, and diagnostic tests for
cancer. Therefore, nano-biosensors are indeed a useful tool in tissue engineering for either
diagnosis or therapy [48,49]. Figure 2 shows the different applications of nanobiosensors.

Figure 2. Advancements and applications of nano-biosensors (reprinted from Ref. no. [2], copyright
2021, MDPI).
Biosensors 2023, 13, 40 6 of 32

Nanosensors, nanoprobes, and other nanoscale technologies have greatly improved

the quality of chemical and biosensing structures. These nanodevices have been developed
with fast reaction times and minimal power consumption in consideration. Electrochemical
indications of biocatalytic processes at the conductive junction have been enhanced with
the help of nanomaterials such as magnetic nanomaterials, metal and oxide nanoparticles,
QDs, metallophthalocyanines, and carbon materials. Functionalized nanoparticles attached
to organic molecules have been produced for their potential application in biosensors.
There is a wide range of methods for synthesizing nanostructured materials and nanode-
vices, while the choice of the best options solely depends on the materials’ properties, the
nanomaterial kind being synthesized (0D, 1D, 2D), the size of the required amount, and
many more factors [50–52]. Especially in biosensing applications, nanostructured materials
are being developed using a wide variety of physical processes [53], including physical
vapor deposition (PVD), high-energy ball mixing, melt mixing, electric arc, laser ablation,
and sputtering.
Nanostructures are also synthesized chemically, most notably by the sol–gel technique
and inverse micelles formation. Materials with nanostructures may be synthesized by
using a bottom-up and top-down technique. The bottom-up approach is the downsizing of
material building blocks associated with self-assembly results to produce nanostructures.
QDs and nanoparticles are created from colloidal dispersions using these techniques. The
lower failure rates and more consistent elemental composition of such methods make them
a suitable alternative [3,54]. Top-down methods, on the other hand, begin with the desired
nanostructure and then carefully design the processing of macroscopic structures. Some
examples are ball milling, extreme plastic deformation, and inductively coupled plasma
(ICP) etching. The existence of very high numbers of flaws in the surface structure is,
however, a fundamental downside of these techniques [55].

4. Architectural Design
In order to design and create a biosensor with good sensing abilities, its architectural
design based on various materials must be considerably taken care of. The use of conductive
polymers (CPs) and transition metal oxides may result in designing a good biosensor with
enhanced capabilities. CPs have recently been synthesized by various synthesis methods
such as biochemical, chemical, and electro-chemical formation techniques [56]. Various
types of research has been completed in bringing out better methods for the synthesis
of CPs which in turn can be used for the design of durable and reliable biosensors. It
has been stated in many studies that selecting the most suitable monomer element for
fabricating a CP layer with sensing capabilities significantly influences the architectural
design of a developed biosensor. CPs have been characterized by the presence of delocalized
π-electrons at the polymer chain backbones which makes the CPs hold some unique
properties such as low ionization potential, enhanced electrical conductivity, and some
other exciting characteristics [57–59]. Such standalone properties have allowed CPs in
various significant applications such as transistors, rechargeable batteries, light-emitting
diodes, electrochromic displays, biosensors, smart windows, and photovoltaic devices.
CPs have been utilized lately to construct various biosensors and catalytic sensors for
bioanalytics. Redox enzyme–glucose oxidase (GOx) is a glucose biosensor in which the
design of the biosensor usually includes the CPs for biological element recognitions. As
stated by various research works on GOx sensors, it can be used as a biocatalyst during the
manufacturing of various CPs including polytiophene, polyaniline, and polypyrrole [60,61].
Certain disadvantages are associated with traditional bioanalytical techniques, such
as the enzyme-linked immune sorbent assay (ELISA), including expensive biomaterials
having to be used, complex analysis processes, and the techniques not being long-lasting,
even though they possess numerous advantages such as accuracy in target biomolecule
detection. In order to overcome this complexity, the use of affinity sensors has been
recommended for various applications. Natural or artificial receptor-based biosensors,
antibody-based biosensors, molecularly imprinted polymer (MIP)-based biosensors, and
Biosensors 2023, 13, 40 7 of 32

DNA-aptamer-based biosensors are some of the classifications of affinity sensors. Amongst

these, MIP-based biosensors are considered to be effective owing to their non-dependency
over expensive biological molecules and they have been built solely based on the polymeric
matrix [62–64]. Transition metal oxides and MXenes have recently been developed (during
the last decade). Two-dimensional materials for the architectural design and construction
of biosensors have been developed owing to their excellent semiconducting properties,
metallic conductivity, and/or a combination of both. Such unique property combinations
can be used for the design and manufacture of biosensors, wearable electronic devices,
and biofuel cells [65,66]. When compared with the physical properties and structural
relationships of typical 2D materials such as graphene, MXenes have been found to possess
similar properties and most of these materials depend on 2D transition metal carbides,
nitrides, and carbonitrides. Owing to the flexibility offered by MXenes towards the design
of biosensors when compared with other materials, they have been proven to be one of the
ideal candidates for the design of biosensors [67,68].

5. Nanomaterials for Biosensors

The evolution of research on biosensors has gained attention among researchers with
its potential utilization in industrial applications and scientific fields including bioinformat-
ics, biotechnology, electronics, materials science, healthcare, medical science, and so on [69].
A biosensor is a device that can sense a biological element in contact with an analyte and
transduce the biological response received into an appropriate signal through a transducer.
There are many challenges in designing biosensors such as sensitivity, response time, repro-
ducibility, and poor detection limits [70]. Among the many challenges in the development
of biosensors, the major concern is the limit of detection (LOD) of biological analytes [71].
The importance of this emerging technology has been intensified to the next level with the
contribution of blooming nanotechnology. Nanobiosensors have recently been a hot topic
of research as nanostructures with their ultra-low dimensions showcase novel properties
over their bulky counterparts. Hence, nanomaterials are being used as candidates for
transducer coatings in order to achieve solicited detection at the picomolar level [72].

5.1. Graphene-Based Biosensors

Nanotechnology praises graphene as a ‘wonder material’ owing to its extraordinary
physicochemical characteristics. Characteristics such as high conductivity, low charge
carrier resistance and a high surface–volume ratio make graphene an ideal candidate for
designing a transducing material (Figure 3). Due to its one-atomic thickness, graphene
is a 2D structural material. It is a sp2 hybridized form of carbon with a hexagonal lattice
structure [73]. The conductivity and dimensionality of graphene sheets can make every
atom easily exposed to environmental fluctuations which is very necessary for a sensing
application [74]. Graphene is reported as the material with the lowest resistivity in the
world, with it having a conductivity of 106 s/m and resistivity of only 10−6 ohm.cm which
is a lot lower than copper [75].
Graphene can be synthesized in a wide number of ways; however, techniques for
synthesizing bulk graphene on a large scale are still lagging which obstructs its utilization in
various potential applications. GO is obtained out of graphene through oxidation through
several routes and GO can be further converted to rGO through the reduction of GO [76].
There are numerous physicochemical methods available to synthesize graphene and its
derivatives which include rubbing graphite on a surface [77], physical exfoliation through
scotch tape [78], the chemical oxidation of graphite [79], the arc discharge method, ball
milling, chemical vapor deposition, and liquid phase exfoliation [80].
Biosensors 2023, 13, 40 8 of 32

Figure 3. Chemical and physical characteristics of graphene and related materials (reprinted with
permission from Ref. no. [73], Copyright 2021, Elsevier).

A few experimenters have designed an ultra-highly sensitive graphene optical biosen-

sor (GOB) for detecting whole living cancer cells responding to the chemotherapy drug
paclitaxel during drug delivery. In their work, a novel optical strategy for detecting re-
fractive index (RI) changes has been followed with the advantage of sensing the living
cells label-free. This method is also beneficial in terms of non-destructive measurements
and dynamic cell monitoring free from electro-magnetic interference and surface con-
tamination [81]. They demonstrated experimentally that the detection of ultra-small RI
changes of 1.35 × 10−7 was made possible using a GOB with a corresponding sensitivity of
1.2 × 108 mV/RIU and a response time of 260 ns. On the other hand, field effect transistor
(FET)-based biosensing technology has been extraordinarily explored for instantaneous
highly sensitive measurements using a tiny number of analytes [82].
Moreover, when compared to optical biosensors, FET-based biosensors eliminate
the complications of enzyme labelling and do not necessitate expensive optical instru-
ments [83]. Afsahi et al. [74] deposited high-quality single-layer graphene by the chemical
vapor deposition (CVD) technique over a commercially available biosensor chip for the
specific detection of an epidemic virus named Zika viral non-structural protein 1 (ZKV
NS1). The deposited graphene layer over the commercial biosensor chip works based
on the field effective biosensing (FEB) technology wherein the channel current and gate
capacitance in the graphene transistor chip shift in accordance with the immobilization
of the biological targets to the gate current. Their experiment with the cross-linkage of
Zika NS1 to a graphene biosensor chip resulted in ZIKV NS1 detection at the very low
concentration of 0.45 nM. A few experimenters have made some serious efforts towards
fabricating a graphene FET-based immunosensor for rapid detection of the COVID-19 spike
protein. In their work, researchers constructed a biosensor by immobilizing two receptor
binding domains such as human angiotensin-converting enzyme 2 (ACE2) and SARS-COV
spike S1 subunit protein antibody (CSAb)—OVID-19 and compared their performance.
They observed that the CSAb-modified biosensor could deliver real-time detection with an
LOD down to a 0.2 pM concentration with a fast response time of two minutes [83].
Similarly, real-time detection of the COVID-19 virus from the nasopharyngeal swab
specimens of affected patients using graphene-based FET biosensors has been reported
by a few researchers (Figure 4). A sensor was designed which could detect the antibody
through the graphene layer coating over the FET biosensor with the SARS-COV-2 spike
protein with an LOD as low as 1 Fg/mL [82]. A novel concept of biological sensing through
electrolyte-gated rGO FETs has been demonstrated by Aspermair and his co-researchers to
Biosensors 2023, 13, 40 9 of 32

identify the E7 protein of the human papillomavirus (HPV) in human saliva. HPV has been
reportedly noted for causing cervical carcinogenesis through sexual transmission. They
have successfully fabricated pyrine-modified rGO-based FET and achieved an LOD of
100 pg/mL (1.75 nM) for HPV-16 E7 [84].

Figure 4. Schematic diagram of COVID-19 FET sensor operation procedure (reprinted from Ref.
no. [82], copyright 2020, American Chemical Society).

5.2. Gold Nanoparticles (GNPs) in Biosensors

GNPs offer an excellent platform in biosensing with their unique colorimetric property
depending on their shape, size, and state of aggregation in the presence of analytes [85].
The transformation of GNPs from a monodispersed state to an aggregate state in a liquid
medium induces a visually evident color change from red to blue. With a diameter between
1–100 nm, GNPs hold a high surface-to-volume ratio and high surface energy. They
facilitate rapid direct electron transport between a wide range of electrodes and electro-
active species. GNPs are thus utilized for biosensing applications in the fabrication of optical
biosensors, electro-chemical biosensors, and piezo-electric biosensors. In optical biosensors,
the changes in the light or photon output are sensed to give the appropriate signal. GNPs
are utilized in optical biosensing based on their optical sensing modality and surface
plasmon resonance (SPR) behavior. GNPs are known to have the ability to enhance the local
electro-magnetic field enabling SPR signal amplification which can bring up a dielectric
constant variation and cause luminescence from the metal–liquid surface [86]. These
distinct characteristics of GNPs have been largely utilized in the biosensors’ fabrication of
food safety applications in detecting various allergens and contaminants (Figure 5).

Figure 5. Schematic overview of different mechanisms of GNP-based biosensors for food safety
detection (reprinted with permission from Ref. no. [87], copyright 2021, Elsevier).
Biosensors 2023, 13, 40 10 of 32

Interestingly, a few researchers have incorporated GNPs into designing micro-cantilever-

based biosensors to detect protein biomarkers. They have embraced a technique of crafting
a microcavity at the free end of the cantilever for a local biological reaction that facilitates
in achieving high detection accuracy. The detection limit attained for the GNP-amplified
biosensor was about 21 pg/mL, which is 70 times of magnitude smaller than the one
without GNP amplification [88]. Yuan’s group have employed GNPs to detect DNA for
allergies to peanuts, soybean, and sesame through colorimetric readouts. During the
process of detection, the allergen gene releases long double-stranded DNA from the GNP
surface and makes them aggregate and change to a blue color with the addition of NaCl.
If the gene sample is below the threshold level, then the DNA probes would stick to the
GNPs inhibiting them from aggregation and remaining in the red color. With this technique,
they were able to recognize allergen gene targets with an LOD of 0.5 nM [89]. Researchers
developed a GNP-based biosensor with the first-ever dual-sensing technology to rapidly
detect SARS-CoV2 spikes using human saliva [90]. The developed system was able to
detect SARS-CoV-2 spike antigens through electrochemical and colorimetric methods. The
colorimetric assay detected SARS-CoV-2 spikes with the aid of antibody–antigen interaction,
and it resulted in the accumulation of GNPs, therefore, changing the color from red to
purple with an LOD of 48 ng/mL. In the electro-chemical detection method, the as-prepared
probe solution is dropped on the disposable screen-printed gold electrode with a very low
LOD of 1 pg/mL [90]. Figure 6 shows the enlarged views of fabricating a biosensor using
the cantilever method.

Figure 6. Enlarged view of the biosensor fabricated through the cantilever method and the process
flow adopted for the manufacturing of the biosensor (reprinted with permission from Ref. no. [91],
copyright 2021, Elsevier).

A few experimenters have demonstrated the real-time detection of hepatitis B surface

antigen (HBsAg) by graphene GNP hybrid biosensors. The GNPs were decorated on
the graphene layer substrate through π–π interaction stacking of single-stranded DNA
(ssDNA). The developed hybrid sensor had the potential to detect HBsAg down to the
detection limit of 50 pg/mL [92].
Biosensors 2023, 13, 40 11 of 32

5.3. CNTs in Biosensors

Carbon in its allotropic form is known as CNT and is distinguishably known for its
excellent mechanical, electrical, electrocatalytic, and thermal properties. It is also expressed
as a rolled-up graphene layer and its properties are determined by the number of composed
walls [93]. The special geometry of CNT has attracted many potential applications in the
field of biosensors. Single-walled CNTs (SWCNTs) have the lowest diameter ranging
between 1 nm to 2 nm, while multi-walled CNTs (MWCNTs) can have diameters up to
100 nm. Due to the large number of layers around them, they possess many inevitable
defects. Double-walled CNT (DWCNT) on the other hand falls intermediately between
SWCNT and MWCNT with many properties of SWCNT as well as a smaller number of
defects when compared to MWCNT [94]. CNTs showcase better biosensing properties
when doped with polyaniline (PANI). PANI is known for its improved redox activity which
when combined with CNT could generate an amplified signal response in electrochemical
biosensors. The CNT–PANI nanohybrid possesses a unique flower-like structure providing
a large surface area and has been experimented for micro bacterium tuberculosis (MTB)
detection [95]. The nanohybrid could deliver super sensitivity of MTB with an LOD of
0.33 fm. Moreover, the electrical properties of CNT have been employed in lactase detection
from human sweat samples. A few experimenters have developed special wearable CNT-
based biosensor-painted gloves for the determination of lactase present in human sweat
as depicted in Figure 7. The constructed CNT/Ag/AgCl painted gloves showed sensitive
detection of lactase up to a detection limit of 1.4 µm [96].

Figure 7. Camera images showing the process of fabrication of CNT-based biosensor-painted gloves
(reprinted from Ref. no. [96], copyright 2018, MDPI).

The utility of CNTs in dermal biosensing has paved the way for cost-effective health
monitoring in a non-invasive manner. Some experimenters have designed a microneedle
array (MNA)-based polylactic acid (PLA)/CNT composite biosensor for thermal injuries.
The matrix of micro-needles with PLA provides outstanding mechanical properties for
skin penetration without causing pain/bleeding and it acts as in situ in electrochemical
biosensors. Electrochemical measurements in ex vivo porcine skin produced a linear current
response corresponding to an LOD of 180 µm [97]. Figure 8 illustrates the process of the
fabrication of biosensors and the morphology of the resulting biosensor surface.
Biosensors 2023, 13, 40 12 of 32

Figure 8. SEM images: (A) conical microneedles fabricated through a micromolding technique,
(B) lateral dimensions of the microneedles, (C) longitudinal dimension of the microneedle, (D) mor-
phology of fractured microneedle along with the stump, (E) enlarged fracture morphology, and
(F) micrographic image showing the complete view of the microneedle mounted on an composite
base (reprinted from Ref. no. [97], copyright 2019, American Chemical Society).

5.4. QD-Based Biosensors

QDs or the so-called fluorescent semiconductor nanocrystals are nanoparticles pos-
sessing diameters less than 10 nm. Recently, they have attracted significant interest in
cutting-edge biosensing technology. The astounding optical, electronic, and size-dependent
luminescent properties of QDs are much utilized by researchers in sensing applications [98].
QDs are proven to offer a high surface-to-volume ratio and extraordinary charge carrier
transport properties that could support improving the performance of a biosensor. In this
aspect, carbon quantum dots (CQDs) have become noteworthy in frontline sensing research
works because of their striking properties such as low toxicity, higher solubility, chemical
stability, and versatility [98,99]. For instance, a biosensor based on CQDs was designed
by Wei’s group for the rapid detection of a carcinogenic element acrylamide (AM) from
food products. CQDs were manufactured through a one-pot hydrothermal technique and
coupled with ssDNA to quench their fluorescence. During the addition of AM, ssDNA
formed a hydrogen bond with AM leaving behind very few free DNAs and eventually
reduced the degree of fluorescence provided by CQDs. The fluorescence property of CQDs
and the high affinity between ssDNA and AM were utilized in the real-time detection of
AM from bread crusts resulting in an LOD of 2.41 × 10−8 M [99]. ZnO QD-based biosen-
sors were developed by Kamaci’s group for the detection of cysteine in solutions. They
came up with a novel strategy of developing a fluorescent probe using melamine-modified
ZnO QDs. The ZnO QD-based fluorescent biosensor displayed a stronger fluorescence
response toward cysteine detection with a linear range from 0.1 to 600 µm and an LOD of
0.642 µm [100]. Figure 9 shows the mechanism of fabricating a fluorescent biosensor for
cancer biomarker detection.
Biosensors 2023, 13, 40 13 of 32

Figure 9. Fluorescent biosensor working mechanism for the detection of cancer biomarkers (reprinted
from Ref. no. [101], copyright 2020, American Chemical Society).

Furthermore, detecting small lung cell cancer rapidly and in a premature state has been
made possible by designing biosensors with graphene quantum dots (GQDs) as energy
donors while the energy receptors were GNPs [101]. The fluorescence response study
carried out for lung cancer detection offered a notable LOD of 0.09 pg/mL with a response
time of 17 min and a larger detection range from 0.11 pg/mL to 1002 ng/mL. A simple and
low-cost textile-based wearable sensor for glucose and H2 O2 sensing has been developed
by some researchers as depicted in Figure 10. A fabric-based nanofilm technology was
developed by integrating Prussian blue (PB) with CdSe QDs and rGO QDs through oil–
water self-assembly engineering over a flexible ITO substrate. The films expressed excellent
electro-chemical sensing activity with a high sensitivity of 53.8 µA mM−1 cm−2 for H2 O2
and 37.24 µA mM−1 cm−2 for glucose, respectively [102].

Figure 10. Schematic of PB–rGO–QDs film preparation through experimental method (reprinted
with permission from Ref. no. [102], copyright 2022, Elsevier).

5.5. Cell-Based Biosensors

Among the biosensors that are being used as analytical tools in medicine, food, bio-
processing, industry, and environmental monitoring, cell-based biosensors (CBB) have
gained substantial attention as they are compact and provide a higher platform for biologi-
cal activity. CBBs use bioactive compounds viz., enzymes, microorganisms, algae, fungi,
bacteria, plant cells, etc., as the analyzer with an appropriate transducer attached to it,
wherein most reported sensors depend on microorganisms such as bacteria and yeast [103].
Luminescent bacteria equipped with reporter protein are used in rapid food safety detec-
Biosensors 2023, 13, 40 14 of 32

tion. The reporter gene exhibiting luminescent or colorimetric properties uses them as the
detecting signal [104].
Fan et al. [105] engineered a Saccharomyces cerevisiae (yeast)-based biosensor for the
on-site detection of copper ion (Cu(II)) (Figure 11). The sensor deliberately gives switch-like
behaviour to respond with ‘yes/no’ for the presence of Cu(II) in the analyte. The sensor can
respond with colorimetric output when integrated with the betaxanthin chromatic pheno-
type and can give styrene-based olfactory outputs when integrated with 2-phenylethanol.
The detection of Cu(II) with betaxanthin-based colorimetric assay showed a limit of detec-
tion as low as 0.32 ppm. However, olfactory output required an intermediate complicated
process of eliminating background odour resulting with 21.0 ± 1.48 mg/L of styrene which
was beyond the human olfactory sensing limit.

Figure 11. Betaxanthin-based biosensor for copper ion detection. (A) Schematic of copper ion
detection, (B) detection using the biosensor phenotype, (C) various water sources with 2 ppm copper
concentrations (reprinted with permission from Ref. no. [105], copyright 2022, John Wiley and Sons).

Micro-level contamination of mercury (Hg2+ ) in water was assessed by employing

a sensor based on bioluminescent Escherichia coli. E. coli strains were implanted within
a photon sensitive small-dimension silicon photomultiplier (SiPM) optical detector [106].
Here, when the sensor encounters the presence of Hg2+ ; the luciferase protein in E. coli is
induced to produce photon instantaneously giving out bioluminescence. An increasing
trend in the bioluminescence was observed for increased concentrations of Hg2+ in the
water sample as shown in Figure 12. Without additional reagent the developed biosensor
was able to provide Hg2+ detection in water with an LOD down to 0.25 µg/L with a
dynamic range of 0.25–25 µg/L.
Biosensors 2023, 13, 40 15 of 32

Figure 12. Photo images of the bioluminescence of the E. coli bacteria in mercury ion-treated water under
different conditions: (a) light; and (b) no light (reprinted from Ref. no. [106], copyright 2019, MDPI).

5.6. COF- and MOF-Based Biosensors

Covalent organic frameworks (COFs) are crystalline materials developed from organic
molecules with nano/microporous structures comprising covalent bonds. COFs are char-
acterized by various advantages such as a completely conjugate structure, huge surface
area, and one atomic thickness dimension. Hence, they find their potential applications
in sensors, electrocatalysis, energy storage, and electrochemical devices [107,108]. Recent
research has focused on the synthesis and design of COFs which belong to the category of
porous crystalline organic nanomaterials owing to their inherent merits such as chemical
and physical durability, excellent structural properties, strong covalent bonds, and high sur-
face area due to porosity. COFs are typically applied in gas sensing, storage, and catalytic
properties along with their use in sensing applications owing to their energy storage and
optoelectronic properties. Utilization of COFs for the fabrication of biosensing applications
was possible due to the short interlayer distance between the 2D structures which exists
between the interactions between aromatic organic molecules within the layers [109–111].
Metal–organic frameworks (MOFs) are the materials with the most exciting archi-
tectures which initiates the fires of recent research through their excellent properties and
applications [112]. MOFs are fabricated through the assemblage of multi-functional organic
linkers and metal nodular clusters and possess a high level of porosity with a large surface
area, and customizable and tunable characteristics. MOFs find most of their applications
in biosensors, drug delivery, gas storage, biomedicines, biotechnology, biocatalysis, and
bioseparation [113,114]. The application of MOFs has recently been extended to biosensing
by a large amount for sensing of various analytes in various fields such as biomedicine,
food, industry, and the environment. Electrochemical sensing has been the most com-
mon application of MOFs owing to their better insulation characteristics. When different
molecules such as enzymes, antibodies, and aptamers are incorporated into MOFs, the
scope of their applications in biosensing fields in the detection of proteins and DNA has
been proven to expand further [115,116].
Biosensors 2023, 13, 40 16 of 32

5.7. Other Nano-Materials in Biosensors

Transition metal oxides have been much explored recently for bio-sensing applications
with their distinct electrochemical properties and variable oxidation states [117]. They are
very well known for their cost-effectiveness and structure-dependent response. Oxygen
atoms bound to the transition metal led to different polymorphs with variable stoichiome-
try [118]. The superior electrocatalytic activity, biocompatibility, and high surface area of
Co3 O4 has been taken up in designing a biosensor for glucose detection. The cubic Co3 O4
crystals were fed as ink to screen-print the biosensing chip circuit. The chip exhibited a
high sensitivity for glucose with a detection limit of 10 µM with a wide range from 10
to 600 µM [117]. TiO2 , one of the most versatile metal oxides, has been effectively used
in biosensor design with its photoelectrochemical properties. Tian et al. [119] reported
the in situ integration of TiO2 nanotube arrays with CdS QDs for the detection of asulam
(pesticide) in real environmental water samples. The fabricated sensor demonstrated a
substantial linear range of 0.02–2.0 ng mL−1 with an LOD of 4.1 pg mL−1 . ZnO nanorods
grown on a SiO2 /Si substrate provided good sensitivity for phosphate. The developed FET
biosensor accomplished a sensitivity of 80.57 µAmM−1 cm−2 in a linear range of 1–7000 µM
with an LOD of ~0.5µM [120]. A polymer-based composite between polyaniline nanosheets
(PANINS) and NiO nanoparticles was prepared to bring out a non-glucose-detecting enzy-
matic biosensor [121]. The invented biosensor possessing a screen-printed NiO@PANINS
electrode established a high sensitivity of 5625 µAmM−1 cm−2 with an LOD of 0.06 µM.
The different nanomaterials used for the biosensor are presented in Table 1.

Table 1. Nanomaterials used in biosensors for the detection of various analytes.

Material Target Analyte Methodology LOD Detection Range Ref.

Graphene ZIKA NS1 FEB technology 0.45 nM - [74]
Graphene Living cells Refractive index changes 1.2 × 108 mV/RIU - [81]
Graphene SARS-COV 2 FET biosensing 1 fg/mL - [82]
Graphene COVID 19 Receptor binding domains 0.2 pM - [83]
rGO E7 protein of HPV Electrolyte gated FET 100 pg/mL - [84]
GNP Protein biomarker Micro-cantilever technology 21 pg/mL - [91]
Gene allergic to peanut,
GNP Colorimetric 0.5 nm - [89]
sesame, and soybean
Dual sensing-
48 ng/mL
GNP SARS-COV2 Colorimetric - [90]
1 pg/mL
Electrochemical
Graphene-GNP Hepatitis-B π-π stacking of ssDNA 50 pg/mL - [92]
Microbacterium
CNT-PAN Electrochemical biosensing 0.33 fm - [95]
Tuberculosis
CNT Lactase Fabric-based wearable gloves 1.4 nm - [96]
CNT/PLA Thermal injuries Microneedle array matrix 180 µm - [97]
CQD Acrylamide Fluorescence 2.4 × 10−8 M - [99]
ZnO QDs Cysteine Fluorescence 0.642 µm 0.1 µm–600 µm [100]
0.11 pg mL−1 to
GQD-GNP Lung cancer Fluorescence 0.09 pg mL−1 [101]
1005 ng mL−1
37.26 µA/mM.cm2
(Glucose)
PB-CdSe QD-rGO QD Glucose and H2 O2 Wearable sensor - [102]
53.8 µA/mM.cm2
(H2 O2 )
Colorimetric and 0.322 ppm and
Saccharomyces cerevisiae Cu(II) - [105]
Olfactory output 21.0 ± 1.48 µg/mL
Escherichia coli Hg2+ SiPM optical detector 0.25 µg/L 0.25 to 25 µg/L [106]
Co3 O4 Glucose Electrocatalytic 10 µM 10–600 µM [117]
TiO2 /CdS QDs Asulam Electrochemical 4.1 pg/mL 0.02–2.0 ng mL−1 [119]
ZnOnanorods Phosphate FET biosensing ~0.51 µM 0.11 µM–7.2 mM [120]
NiO@PANINS Glucose Amperometry 0.06 µM - [121]
Biosensors 2023, 13, 40 17 of 32

6. Applications of Nanotechnology-Based Biosensors

The development of biosensors has reached a tremendous altitude in various fields
such as healthcare, bio-medicine, and food processing. The progression in nanotechnology
with the development of various nanomaterials and nanotechnology goes hand in hand
with the advancement of novel biosensors [122]. The ability of nanotechnology to control
and regulate materials at their atomic and molecular level has opened avenues for a better
understanding of their fundamental properties. The dimensionality of any material plays
a vital role in determining its physicochemical and biological properties which assist
in adopting them even in multidisciplinary applications [123]. The evolution of nano-
biosensors set forth their application in biomedical diagnosis for monitoring and detection
of the ultra-low concentration of analyte and the physicochemical phenomenon even in
unreachable parts [124]. Biosensors are of different types based on the nature of their vital
components such as bioreceptors and transducers.
Bioreceptors are the primary component of a biosensor based on which they are
classified as enzymatic biosensors [125], immunosensors [126], aptamer biosensors [127],
and microbial biosensors [128]. Secondly, based on transducer type, they are categorized
as electrochemical biosensors [129], thermal biosensors [130], electronic biosensors [131],
optical biosensors [132], and gravimetric biosensors [133]. Furthermore, based on the
detection technology they are classified into nano-biosensors, surface plasma resonance
biosensors [134], biosensors-on-chip [135], and electrometers [69]. With the remarkable
progress established in the field of nanotechnology, biosensors will reach their pinnacle in
biocompatibility, selectivity, sensitivity, wearability, and LOD in the upcoming years [136].
A few of the applications of biosensors are elaborated on in the following sections.

6.1. Biomedical Applications

The assessment of the progression in the health of patients is very crucial and needs
precise monitoring of the condition of the disease to minimize the rate of mortality. Biosen-
sors play a significant role in the biomedical field through multiple applications such as
wearable devices, implantable devices, and so on to define the treatment protocol for the
speedy recovery of patients. The bioresorbable pressure sensor designed by Shin et al.
investigates the healing process of chronic disease and the pressure sensor is shielded by a
SiO2 protective layer [137]. On the other hand, healthcare electronics are ruling the world
presently. Sheng et al. fabricated a wearable flexible capacitor biosensor with many biomed-
ical aspects such as accident alert, pressure measurement, remote control, and motion
feedback [138]. Biodegradable polymers have emerged as a new class of biomaterials com-
patible with sensing applications. A few experimenters have manufactured an implantable
biosensor for detecting glucose that could sense glucose catalytic reactions in a range of
0–10 mM [139]. Biosensors have created an optimistic revolution in the biomedical field.
The specific biomedical applications of biosensors are discussed in the upcoming sections.

6.2. Cancer and Bone Disease

Cancer biomarkers (CBs) such as DNA, RNA, proteins, enzymes, and hormones that
are released by genetic alteration are used as the essential aspect for screening the status of
cancerous cells in patients. Detection of these CBs in the human body helps in undertaking
relevant therapy with point-of-care applications [140]. Conventional techniques in practice
for the detection of CBs impose technical limitations and require replacement with novel
technology which is economically feasible and technically sound [141]. Premature cancer
detection could highly reduce the rate of mortality and hence, it requires a highly precise
tool for biomarker detection. Therefore, biosensors with their overwhelming properties
have been advocated for cancer detection primarily in the last decade. According to
Hasan et al., cancer-detecting biosensors are categorized as per the type of transducer
and biological response as optical, mass-sensitive, and electrochemical sensors. Figure 13
depicts the workings of biosensors for cancer biomarker detection.
Biosensors 2023, 13, 40 18 of 32

Figure 13. Schematic of biosensors’ working principle for cancer biomarkers’ detection (reprinted
from Ref. no. [140], copyright 2021, Elsevier).

The enhanced biosensing by graphene through its property of easy binding with
carbon-based rings present in biomolecules has been integrated with TiO2 for early-stage
cancer detection [142]. This multilayer plasmonic sensor exhibited sensitivity for cancer
cells from the skin, cervix, blood, adrenal glands and breasts with maximum angular
sensitivity of 282.86 deg/RIU. Kim et al. [143] utilized Raman scattering as a tool for
detecting breast cancer from human tears. This was realized by fabricating substrates
with a gold-decorated, hexagonal-close-packed polystyrene (Au/HCP-PS) nanosphere
monolayer and detection with a portable Raman spectrometer. In the same grade, bone
diseases such as osteoarthritis and rheumatoid arthritis emphasize the essentiality of
early diagnosis and treatment to elucidate the level of joint damage and inhibit added
degradation of articular cartilage [144]. Hu et al. [145] proposed an SPR-based biosensor
that could sense the miR-15a biomarker for the identification of rheumatoid arthritis with
an LOD of 0.56 fM in a linear range of fM-0.5 nM. Similarly, a poly-hydrogel-film-based
electrochemical biosensor was reported for the detection of osteoarthritis with an LOD of
2 nM with a linear range of 2–2000 nM [146].

6.3. Tissue Engineering Applications

The tissue engineering field is a versatile approach that could help in replacing dam-
aged tissues involving the main biological components such as cells, scaffolds, and stimula-
tory molecules. This technology is very well received in terms of cost-cutting in comparison
with organ/tissue transplantation [147]. The potential utilization of biosensors for tissue
engineering and regenerative medicine is very minimum. In general, biosensors are used
in the field of tissue engineering to ensure proper tissue growth by monitoring critical
parameters such as oxygen/nutrient uptake and the release of metabolites [148]. An overall
outlook on the usage of biosensors for the investigation of the quality of 3D constructs is
explicitly shown in Figure 14. Because of the small dimension of biosensors, they can be
relatively designed in desired shapes according to the cellular environment and can also be
inserted inside tissues and organs. A platinum-wire-based enzymatic biosensor has been
proven to be biocompatible and helpful in monitoring the quality of tissue implants when
inserted into brain tissues [149]. Electrochemical sensors are useful for in vitro applications
in monitoring the redox compounds that are released from the implants’ post-biological
insertion. Indeed, in recent years, amperometric sensors have been applied in both in vitro
and in vivo applications to analyze the quality of 3D constructs by sensing the release of dif-
Biosensors 2023, 13, 40 19 of 32

ferent compounds such as dopamine [150], ascorbic acid [151], glucose [149], oxygen [152],
and nitric oxide [153].

Figure 14. Applications of biosensors in the tissue engineering field using (a) electrochemical tech-
niques, (b) the optical method, (c) real-time monitoring of quality, and (d) longitudinal studies
(reprinted from Ref. no. [148], copyright 2021, Springer Nature).

6.4. Microfluidic Systems

Microfluidic systems offer manipulation of sensing through a small volume of fluids
that pass through lithographic microchannels. These lab-on-chip (LOC) devices can mimic
the microenvironment around the conventional cell culture and help to isolate microparti-
cles [154]. A biosensor integrated with a microfluidic system was designed for the diagnosis
of breast cancer through the identification of an extracellular vesicle microRNA biomarker.
The fabricated LOC captured breast cancer biomarker microRNA with a detection limit of
84 aM with range of 1 fM to 1 nM [155].
A unique opto-microfluidic sensing platform was designed by a few researchers for
detecting the COVID-19 virus at an early stage using human plasma, as shown in Figure 15.
The sensing system was able to detect the COVID spike protein with a limit of detection
of 0.5 pM and it consumes 30 min of time to complete the sample analysis [156]. An attempt
to improve the sensing capability of an optical sensor was made by combining the merits
of QDs with microfluidic technology. Two subtypes of the Influenza A virus (H5 and H9)
could be identified by conjugating the antibodies with QDs. A bright fluorescence was
produced by the QD-conjugated antibodies with an LOD of 0.26 and 0.017 HAU for H9
and H5, respectively [157].
Biosensors 2023, 13, 40 20 of 32

Figure 15. The opto-microfluidic chip with gold nanospikes (reprinted with permission from Ref.
no. [156], copyright 2020, Elsevier).

6.5. Diagnostics
Biosensors hold huge potential for real-time microbial diagnostics. Biosensors when
integrated with nanotechnology facilitate rapid, real-time, and accurate detection of molec-
ular biomarkers in real samples. Researchers have focused on designing body-worn
monitoring devices to acquire real-time diagnostic information using different label-free
and lab-on-a-chip bioelectronic systems. A few experimenters constructed a stretchable
electrochemical immune biosensor for the detection of tumor necrosis factor (TNF)-α which
aids in wound heal monitoring. TNF immobilization was carried out using the differential
pulse voltammetry method. The immunosensor shows a sensing performance with a
clinical concentration range of (0.11 pM–0.09 µM) in human serum [158]. The contribu-
tion of electrochemical biosensors in detecting pathogenic bacteria has been elaborated
by Karbelkar et al. [159]. The presence of E. coli in drinking water was detected by an
electrochemical immune biosensor using a screen-printed electrode on a gold substrate
with an LOD of 30 CFU mL−1 [160]. Rapid sensing of many pandemic and epidemic
viruses using biosensing technology has been trending recently. Along the same line, a few
researchers have developed a glycol nanoparticle-based immunosensor for the detection
of the human influenza virus through SPR [161]. The study on the point-of-care detec-
tion of the Ebola virus in human survivors was undertaken by Brangel et al. [162]. The
developed smartphone-based assay for the detection of IgG antibodies on infected patients
in Uganda demonstrated 100% sensitivity and 98% specificity compared to the standard
whole antigen.

6.6. Porphyrin and Phthalocyanine

Porphyrins (Pp) and pthalocyanines (Pc) are a special class of macrocyclic organic
molecules possessing unique photophysical properties and hence find application as flu-
orescence probes in biomedical sensors, drug delivery, and fluorescence imaging. Their
distinct optical properties originate from the arrangement of 18π electrons packed in a
highly conjugated system. They have strong absorption towards the light where Pc absorbs
mostly in the far infrared region [163]. Relaxation between different vibrational levels
induces π–π* transition between the electrons in Pp giving rise to two types of fluorescence.
The first type of fluorescence (S1-fluorescence) is observed in the range of 550–800 nm
and is comparatively stronger than the second type. The other type (S2-fluorescence) of
luminescence is weaker and observed between 400–550 nm [164]. The fluorescence property
has been applied as a promising tool in various biomedical diagnoses. In this regard, a few
experimenters fabricated a carboxyl Pp-based fluorescent biosensor to detect aflatoxin B1
present in medicines. Under optimization, the carboxyl Pp connected to the antigen could
detect a least a value of 8.38 fg mL−1 in a linear range of 100 fg mL−1 to 100 ng mL−1 [165].
Furthermore, Attia’s group used the luminescence property of Pc for the determination
of ovarian cancer antigen CA 125 in human serum samples. The biosensor designed with
Biosensors 2023, 13, 40 21 of 32

Ni-phthalocyanine doped in a polystyrene matrix was able to identify the cancer antigen as
it quenched the intense fluorescence at 790 nm when excited with 685 nm. The investigation
resulted in a detection limit of 1.0 × 10−4 U mL−1 in a linear range of application of about
1.0 × 10−2 −127 U mL−1 [166]. A porphyrin–Co9 S8 nanocomposite synthesized via the
hydrothermal route was applied for the detection of H2 O2 through colorimetric sensing
resulting in an LOD of 6.803 µM in a linear range of 7–100 µM [167].

6.7. Detection of Glucose

Biosensors have become a significant tool in the real-time clinical analysis of tahe wide
variety of molecules present in bio-fluids, most significantly glucose. Glucose level sensing
is essential for monitoring several complications such as diabetes, heart disease, high blood
pressure, and kidney failure. Conventional sensing methods include the usage of enzymes,
such as glucose oxidase, which has several limitations such as being highly expensive,
having poor stability, and low reproducibility [167,168]. Hence, significant exertions have
been made to develop a non-enzymatic electrochemical-based sensors. Ahmad et al. [169]
proposed a novel CuO-nano-leaves-based electrochemical biosensor for glucose sensing.
CuO nano-leaves were synthesized via a facile hydrothermal route and modified with a
glassy carbon electrode. This non-enzymatic biosensor electrode displayed good stability
and reproducibility with a high sensitivity of 1467.32 µA/(mM cm2 ) with a detection limit
of 12 nM in a linear range of 0.005–5.89 mM. A gold-decorated hierarchical Cu nanoflower
coated on GO nanofibers used as an electrode in the electrochemical biosensor (as shown in
Figure 16) showcased excellent electrocatalytic properties in converting glucose to gluconic
acid. During the experiment, the glucose concentration and current increased linearly along
with each one facilitating the monitoring of glucose levels in the biofluids resulting in an
LOD of 0.018 µM in a wider linear range of 0.001–0.1 mM [170].

Figure 16. Schematic of Cu–nanoflower@AuNPs–GO nanofiber-based electrochemical nano-

biosensor fabrication for glucose detection (reprinted with permission from Ref. no. [170], copyright
2020, Elsevier).

A colorimetry-based biosensor with nanolayered manganese–calcium oxide mimick-

ing the enzymatic behavior of glucose oxidase was engineered by Rashtbari et al. [171].
Colorimetric strategy was used for the on-time simultaneous detection of glucose and H2 O2
from a human serum sample. The oxidation reaction taking place to convert glucose into
gluconic acid in the presence of O2 and MnCaO2 changes the color of the reaction solution
to red-orange facilitating the naked-eye detection of glucose by spectrophotometry. The
experiment resulted in the identification of glucose with an LOD of 6.12 × 10−6 M in a
linear range of (0–82.3) × 10−6 M.

6.8. Detection of DNA and Protein

Biosensors are used as an analytical tool for the detection of biomarkers such as DNA
and proteins. In a biosensor, the resultant signal from the reaction of biological components
Biosensors 2023, 13, 40 22 of 32

is converted to measurable signals by a transducer. These biological components are usually

protein biomarkers that underlay the existing condition of a disease. A few experimenters
have manufactured a label-free and sensitive optical biosensor with Si nanowire to detect
C-reactive protein (CRP) present in the human serum. Here, the surface of the nanowire
was modified with a CRP antibody and the LOD for the immunosensor was achieved
down to 1.6 fM [172]. Pork content present in the edible items was detected by a gold–
DNA-conjugated electrochemical sensor [173]. The sensor detected the existence of pork
in food by recognizing the presence of Sus scrofa mtDNA (pig DNA) in various raw and
processed meat samples with an LOD of 0.59 µg/mL. Some experimenters stated the design
of a biosensor for the first time to detect the TAR DNA binding protein, a biomarker for
neurodegenerative disorders. The bioconjugation method for the fabrication of biosensors
helped in cutting down the cost and gave rapid detection of DNA in a detection time of
1 h [174]. The nanomaterials used for various biosensing applications are compiled and
listed in Table 2.

Table 2. List of nanomaterials used for various biosensing applications.

Nanomaterial Applications Target Analyte Methodology LOD Detection Range Ref.

Si nanowire Cancer detection 8-OHdG Electrochemical 1 ng/mL - [175]
GO Cancer detection PEAK1 Electrochemical 10 pg/mL - [176]
Surface Plasmon
TiO2 /Au/graphene Cancer detection MCF7 292.86 deg/RIU - [142]
Resonance
Surface enhanced
Au/HCP PS Cancer detection 2-NAT 100 fM 10−8 –10−3 M [143]
Raman scattering
Rheumatoid Surface Plasmon
SPRi-Gold chip miR-15a 0.56 fM 5 fM–0.5 nM [145]
arthritis Resonance
Poly hydrogel film Osteoarthritis Proteases Electrochemical 2 nM 2–2000 nM [146]
Aptamer
Silver nanoparticle Kidney tissue MCF-7 - - [177]
conjugation
Graphene/gold Liver metastasis PHD-1 Electrochemical 4 µM - [178]
Periodontal
nMgO - GTR - - [179]
tissue regeneration
Zeolite Neuro transmitter Dopamine Amperometry - - [150]
Nanoporous gold Fungus detection Ascorbic acid Electrochemical 2 µmol L−1 - [151]
Carbon Brain tissue Oxygen Biotelemetry ≤5 M - [152]
- Cancer detection microRNA microfluidic 84 aM 1 fM–1 nM [153]
Gold nanospike COVID-19 Cov 2 spike microfluidic 0.5 pM - [154]
H5 0.016 HAU (H5)
- Influenza A microfluidic - [155]
H9 0.25 HAU (H9)
Ni-Phthalocyanine Cancer detection CA125 Optical sensing 1.0 × 10−4 U mL−1 - [163]
Co9 S8 -Porphyrin H2 O2 detection - Colorimetry 6.803 µM 7–100 µM [166]
NiO Glucose detection Saliva Electrochemical ~84 nM ~5 µM to 0.825 mM [168]
CuO nanoleaves Glucose detection - Electrochemical 12 nM 0.005–5.89 mM [169]
Au/Cu/GO Glucose detection - Electrocatalysis 0.018 µM 0.001–0.1 mM [170]
MnCaO2 Glucose detection Human serum Colorimetry 6.12 × 10−6 M (0–82.3) × 10−6 M [171]

7. Challenges and Prospects

In terms of the difficulties associated with translating in vitro systems to in vivo sys-
tems, nanobiosensors are now a fundamental aspect of research. The interconnectedness
of the system’s parameters has a profound effect on the expression of different analytes,
making systematic monitoring in real time an absolute need. Many industries are taking
advantage of recent breakthroughs in the field of nanobiosensors owing to their excel-
lent magnetic, electrochemical sensors, acoustic, piezoelectric, and optical properties, but
tissue engineering has yet to overcome the significant barrier of incorporating these sen-
sors [41,42,53]. No one can deny nanotechnology’s impact in propelling biosensors to new
heights. It has been demonstrated that using nanomaterials/nanostructures for biosens-
ing applications can increase important sensor properties, including LOD, precision, and
reliability. Such cutting-edge biosensors have been shown to rapidly detect the target
analyte, exhibit single-molecule detection, and considerably boost transduction outputs.
The obstacles to biosensors’ widespread use have been eased thanks to these characteristics.
However, there are also drawbacks to this technology, including the unavoidable emission
Biosensors 2023, 13, 40 23 of 32

of nanoparticles further into the atmosphere [84,94]. In addition, quantum effects produce
exceptionally high sensitivity, random noise, and background signals. The response of such
sensor exposure can lead to certain analytes that have been observed to be cross-sensitive,
nonlinear, and unpredictable. Materials such as graphene are promising for biosensing
applications; however, they have not been effectively mass-produced. The world will not be
able to fully use nanotechnology’s incredible potential in biosensors unless these issues are
resolved [104,121]. Figure 17 shows the timeline of developments in the field of biosensors.
Development began with the advent of oxygen-based biosensors and recently QDs have
been used for the development of biosensors. This development timeline is expected to
enter into the field of tissue engineering and other allied biomedical fields where the advent
of nanobiosensors might make a larger difference in comparison to the current system of
biosensors [180,181].

Figure 17. Biosensor development timeline (reprinted from [180], copyright 2022, MDPI).

The use of ML represents one of the innovative technologies that is currently being
applied to the problem of such shortcomings. When considering biosensor implementa-
tions, ML could be considered as an algorithmic strategy for examining sensor data and
determining usable information via statistical methods [145]. Traditional applications of
ML have been in the areas of classification and regression. Naturally, then, such resources
are of great use in the discipline of chemometrics. Support-vector machines, random
forests, artificial neural networks, convolutional neural networks, Naive Bayes, and k-
nearest neighbors are a few of the most popular machine learning algorithms a few of the
most popular machine learning algorithms in use for this purpose. Diverse researchers
have elaborated [146] on the deeper perspectives of using ML algorithms for biosensing
applications and their data processing. Their superior pattern recognition capabilities and
machine learning algorithms, with their superior pattern recognition capabilities, can help
nano-biosensors draw insights from raw data. Examples of applications of such algorithms
include the classification of raw sensor data and the mitigation of cross-sensitivity and
misclassification. Reduced detection limits are possible thanks to the application of ML
algorithms for filtering out irrelevant data from the sensor output.
Biomarker methods will investigate the escalating possibilities for developing targeted
therapeutics, diagnostic tools, and medical equipment. There is room for innovative change
in the way human samples are gathered. Surgically implanted biosensors may play a
significant role in hastening the development of individualized therapeutics. They will
let scientists keep a close eye on the outcomes of potential new treatments in the body,
Biosensors 2023, 13, 40 24 of 32

allowing them to more properly gauge whether a drug can proceed to clinical testing. In
addition, biosensor chip technology can be implanted to detect complex DNA alterations
in the blood before the onset of illness symptoms. Biosensor innovation can be utilized in
reversible and low-cost care point devices. It also has the capability to monitor implanted
devices in real-time. Smart bracelets included in wearable devices can non-invasively
monitor collected samples such as saliva and expelled condensing breath and invasively
collected samples such as blood and interstitial fluid [151,160].
There are several important technical issues that must be resolved, such as extending
the lifespan of the sensors. Biosensors provide a mechanistic understanding of biological
structures down to the molecular level. Numerous fields, including the analysis of biologi-
cal processes, agriculture, medicine, and environmental technology, have found numerous
applications for these kinds of analysis instruments. As a result of this understanding,
numerous approaches have been developed for identifying biomolecules; they serve crucial
roles in many areas of biotechnology, including drug discovery and targeting, pathogen
detection, gene therapy, and many others. The need for biosensors is increasing because
of their widespread application in healthcare and medicine. In addition, advances in
biosensors’ use across various fields—including human health management, patient health
surveillance, diagnosis, and illness detection—have paved the path to rapid expansion in
this field [53,58,124].
Another possibility is wearable biosensors in conjunction with ML for health moni-
toring. On account of their immense potential for the non-invasive evaluation of human
physiology in a broad range of biological fluids, wearable biosensors have garnered con-
siderable attention. Wearable biosensors’ goal is to continually monitor biomarkers by
integrating a succession of sensing devices on flexible patches. Multiplexed sensory data
can be analyzed using ML to determine a patient’s health status from time series data. For
these uses, ML must be transparent. Healthcare providers and policymakers must be able
to grasp the algorithm’s verdict. Meanwhile, a deep training process must have transparent
human knowledge with reasoning principles transparently integrated into it to control and
enforce its learning and decision-making. Furthermore, the size of the samples needed for
ML algorithm training can also be drastically reduced with the help of manual overriding.
This highlights the critical necessity to integrate explainable ML in the field of wearable
electronics in healthcare applications and related medical actions [37,168].

8. Summary and Conclusions

Nanotechnology-enabled biosensor technology has been comprehensively discussed
in the above sections. ML techniques can enhance the quantitative forecasting of trace
analytes, and qualitative discrimination of complicated overlapping signals can also be
accomplished. Deep learning techniques such as convolutional neural networks (CNNs)
and recurrent neural networks (RNNs) in sensory data analysis are specifically witnessing
a rise. Regression analysis with traditional data requires a different formula to determine
the sample’s dependent variables. Typically, there are fewer than two features used as
inputs. In contrast, state-of-the-art ML models can handle a database with hundreds of
input data. A large enough data set is required for deep learning techniques to work.
Researchers can get beyond the data bottleneck between ML and biosensors by designing
and using multiplex or high-throughput biomaterial sensors such as microarrays and
channel fluidic chips.
Biosensors have many uses in the medical profession and are of great benefit to both
patients and clinicians for a variety of reasons, including disease prevention and manage-
ment, clinical diagnosis and treatment, access to health records, and evaluation of treatment
outcomes. Nanomaterials have recently demonstrated extensive use in the creation of
biosensors. The primary goal of clinical medicine is to classify patients into different
categories using simple biosensors. There is widespread consensus that biosensors make
possible tailored medicine possible, which represents a radical departure from conventional
medical practice. This method has profoundly impacted the healthcare industry, which has
Biosensors 2023, 13, 40 25 of 32

led to numerous therapeutic and diagnostic options. Many studies’ findings point to LC
interface concepts that could be applied to constructing stimuli-responsive materials for
highly precise biosensors. The target molecules, immobilization methods, and enzymes,
chosen will all play a role in how fruitful this field proves to be.
Several smart nanostructure-based techniques could be implemented to boost the LC
biosensor’s sensing performance—particularly its low detection limit and high sensitivity.
Moreover, such sensing systems are inexpensive since they can be easily fabricated without
the need for costly laboratory equipment, they require little operational power, and their
sensing results are consistent and repeatable even when performed on different batches.
This article has summarized the methods and mechanisms used to create portable LC-based
biosensors for label-free detection of a specific analyte at a low concentration. The authors
are committed to complete further research into using such sensors for illness diagnosis,
food safety, and the control of epidemics. We think that nano-enabled LCs-biosensors have
the potential to be used for recognizing any biological substances because of their desirable
sensing performance.

Author Contributions: Conceptualization and methodology, M.R.; validation and formal analysis,
R.J.; investigation and resources, C.D.; data curation, L.R.; writing—original draft preparation, R.J.
and C.D.; writing—review and editing, M.R. and L.R. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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