Nanofibers as Efficient Platforms in Biosensing Devices: Comparison
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Please note this is a comparison between Version 3 by Jessie Wu and Version 2 by Jessie Wu.

Biosensors are analytical tools that can be used as simple, real-time, and effective devices in clinical diagnosis, food analysis, and environmental monitoring. Nanoscale functional materials possess unique properties such as a large surface-to-volume ratio, making them useful for biomedical diagnostic purposes. Nanoengineering has resulted in the increased use of nanoscale functional materials in biosensors. Various types of nanostructures i.e., 0D, 1D, 2D, and 3D, have been intensively employed to enhance biosensor selectivity, limit of detection, sensitivity, and speed of response time to display results. In particular, carbon nanotubes and nanofibers have been extensively employed in electrochemical biosensors, which have become an interdisciplinary frontier between material science and viral disease detection. 

  • DNA
  • sensors
  • nanofibers

1. Introduction

The modern world has seen tremendous advancements in technology, which have resulted in decreased mortality rate, among other benefits; yet, this has occurred with a trade-off: the emergence of new health-related problems [1]. Modern society has ironically been riddled with ever-emerging new strains of viruses that have wreaked havoc on the human population, one of which we are witnessing in our life time is COVID-19, which keeps mutating and striking with more lethality. Producing antibodies against any disease is one of the strongest mechanisms that a body can offer. Antibodies or hormones are released against a disease into the body, which act as biomarkers for accurate, reproducible, and objective measurement of disease by establishing a patient relationship with a clinical end point [2]. The clinical end point may include infection, stroke, cancer, heart disease, HIV, etc., which is supported by epidemiological, physiological, pathological, and therapeutic evidence [3][4].
Contemporary society has been more focused on health and the treatment of diseases. Biosensors have evolved over the years owing to advancements in nanotechnology and the understanding of molecular self-assembly and nanoscale principles. The interaction of a bioreceptor with a bioanalyte results in a transduction signal that is fed to detector for a suitable response to be displayed. Hence, biosensors based on their transduction signals can be classified as electrochemical, piezoelectric, field-effect-transistors, thermometric, and optical detection biosensors [5][6]. Biosensors, especially as electrochemical sensors, have been in demand due to their quick detection and diagnosis of disease using specific biomarkers in the human body. Here, the change in electrical properties of the analyte of interest in terms of ions or electrons is detected as an electrochemical signal due to the electron transfer process, which does not require expensive equipment, as is required for acoustic or optical biosensors [7]. The electrochemical detection method, however, requires high sensitivity and a short range of detection; yet, the detection range has so far been particularly large. This can be improved by modifying the surface of the sensors with molecules such as DNA, enzymes, carbon-based nanomaterials, nanoparticles, and hybrid materials [8][9][10][11]. The oxidative DNA damage caused by pharmaceuticals can be investigated with biomarkers, such as 8-oxoG and 2,8-DHA, that show enhanced signals on glassy carbon electrode when modified by DNA [12]. Similarly, significant work has been carried out by using carbon-based nanomaterials as sensing platform for enhanced efficiency and reaction specificity [13]. Many studies refer to improving the sensing ability by modifying the electrode surface with molecules that lead to an improved limit of detection (LOD) by enhancing the surface area and sensitivity of the sensing matrix for the analyte of interest [14][15][16].
Nanomaterials have been found to confer unique properties to sensors by modifying the transduction surface. Nanomaterials typically have at least one dimension that lies between 1 and 100 nm and possess unique physical and chemical characteristics. For instance, due to the small size of nanomaterials, their electrical conductivity and optical properties significantly vary compared with those of their bulk counterparts. These nanomaterials are effective in stabilizing the platform for sensing biological molecules by offering increased surface area, high reactivity, and biocompatibility, as has been discussed in [16][17][18]. Having a high surface area to improve the sensitivity of biosensors along with improved accessibility of the molecule is imperative. One-dimensional nanomaterials such as nanofibers may include synthetic or natural polymers (cellulose), metal-oxide-based nanowires (NWs), carbon nanotubes (CNTs), and carbon nanofibers (CNFs); however, they have not been widely used in the literature for the electrochemical diagnostics of biomarkers. They are promising materials for electrode modification owing to of their structure, which supports efficient electron transport. The conducting features of CNTs are further improved by the incorporation of metal nanoparticles (M NPs). Biocompatible nanomaterials such as iron, gold, zinc, copper, and silver M NPs bestow unique electrochemical recognition capacity to the electrode for the detection of organic analytes. CNTs and CNFs have shown more promise as they offer a long electron pathway and facilitate electrolyte penetration due to their porous nature [19][20], thus improving sensing properties. Nanowires are characterized by a slender structure with excellent electron transport properties, having an aspect ratio (ratio of length to diameter) of 1000 nm, which is smaller than that of nanotubes, which can be used as nanorobots and as microdialysis probe for the detection of glucose in the body [21][22][23]. CNTs have a very high surface area, a higher aspect ratio, and excellent absorption properties. In CNTs, the graphite layer runs parallel to the inner hollow tube; in CNFs, the graphite layer forms an angle with the inner tube axis that can be hollow or solid. This renders the diameter of the CNF to be 10–50 nm; in comparison, CNTs have a diameter of around 100 nm. CNFs have graphite at their edge planes, making the surface highly attractive for the functionalization of materials to form hybrid nanomaterials [24][25]. Electrospinning has been the favored method to synthesize CNFs. It employs the bottom-up approach to form continuous and stable nanofibers (NFs) at low cost, while the chemical vapor deposition (CVD) and plasma-enhanced CVD methods have also been adopted as bottom-up approaches. In electrospinning, optimizing the process and solution parameters results in the controlled morphology of the desired NFs [26][27].

2. Nanofibers as Efficient Electrochemical Sensing Platforms

NFs are tiny1D strands of high-surface-area material with unique highly directional strength and flexibility. These NFs have long been under development; yet, they did not gain much popularity until 1990 with the breakthrough advent of nanotechnology. These are synthesized by different techniques employing top-down and bottom-up approaches. The top-down approach utilizes bulk materials to fabricate NFs and includes chemical and mechanical methods such as grinding, milling, etc. The bottom-up approaches, which are widely used to build NFs from basic building blocks, include techniques such as melt blowing, freeze drying, self-assembly, template synthesis, phase separation, chemical vapor deposition, etc. Electrospinning is the commonly used bottom-up technique, which allows control of the NF morphology for achieving maximum performance by controlling the flow rate, concentration of the polymer solution, voltage, distance between the nozzle and collector, etc. Further descriptive details of the techniques to synthesize NFs are described in [28]. A wide variety of polysaccharides, such as alginate and chitosan; and polymers, which can be natural or synthetic, such as collagen, keratin polylactic acid, poly(lactic-co-glycolic acid) (PLGA), composites, and metals/metal oxides, have been used for the synthesis of NFs. Because of their promising and tunable properties, NFs are perfect candidates for biomedical applications. Electrospun NFs are characterized by a high surface area to volume ratio, which allows for more efficient and sensitive detection of analytes. This is because a larger surface area increases the likelihood of analytes binding to the fiber surface, leading to better sensitivity in detection.
Thus, to harness NFs as an efficient platform in biosensing devices and to further optimize the biointerface for enhanced activities, bioreceptor binding event/interactions should be boosted for an accurate, stable, and fast response time, and sensitive analytical performance. To meet this end, special attention has been focused on lab-on-chip and paper-based sensors as point-of-care (PoC) devices, which are used in the healthcare sector for near-patient testing, deployed irrespective of place and time. PoC is an emerging tool for quantifying and diagnosing biomarkers. However, the commercialization of such devices is tedious, and scientists are striving to develop sensors that can be readily used for applications with the minimum expenditure of resources. Here glucometers and pregnancy tests are some of the examples of on-site self-monitoring of health and have been successfully commercialized.
The electrospinning technique is one of the most commonly used methods to synthesize NFs. The vertical and horizontal set ups in electrospinning result in different electrochemical spun NFs such as single NF and/or NF mats [29][30][31]. These NFs are synthesized by using a range of natural fillers or synthetic polymers, mostly incorporating fillers, nanoparticle (NPs), metal salts, and carbon-based nanomaterials for advanced functionalities. A detailed review on electrospinning can be found in [29][32][33][34]. NFs have a variety of morphologies that are produced by modifying the condition or set up for electrospinning [35]. However, for sensing applications, in order to facilitate the interaction of biological molecules by entrapping them in NF that avoids direct contact with organic solvents, a core–shell NF was devised by using spinneret configuration [36], where injection speed tuned the inner–outer sheath size and thickness. The binding of bioreceptors such as enzymes, DNA strands, antibodies, aptamers, etc. [37][38][39][40][41], to the NFs is carried out by physical or chemical means to induce effective interfacial interaction while retaining the functionalities of hybrid bioreceptor–NF assemblies for detection of analyte of interest. Thus, their superior performance can be attributed to the synergistic role of bioreceptors and NFs.
In order to keep the environment inert for biomolecules, the native structure should be modified in such a way that the reactivity and recognition events are not compromised. Hence, hydrophilic poly(ethylene terephthalate) (PET) and poly(vinyl alcohol) (PVA) have been extensively employed for the purpose; however, to maintain operational stability, they must be further treated by linking to glutaraldehyde (GA), which additionally improves the biocompatibility. Post-treated low-water-soluble NFs can be generated by attaching a hydrophilic polymer/metal oxide, which helps with binding the bioreceptor. Enhanced transduction signals have been achieved by electrospinning NPs/CNTs into nanospun fibers, which improves the electron transfer. For biosensing molecules, NFs have been synthesized by using metal oxides(MO)/carbon/polymers that can be doped with NPs and CNTs for improving conductivity or covered with a conductive layer of polymer [37][42][43][44][45].
These bioreceptors are immobilized on the surface of electrode either by absorption [37][46] or covalent bonding when activated with EDC/ NHS [38][47], or Nafion/chitosan/poly (3,4-ethylene dioxythiophene)polystyrene sulfonate (PEDOT:PSS) entrapment [42][43][48], crosslinking with GA [49], or electrostatic interactions [50], which can detect different analytes of interest. CNFs facilitate electron transfer rate due to their conductive nature and support the adsorption of analytes during the preconcentration step of electrochemical sensing. Furthermore, they have different specificities for different organic analytes, thereby improving the discrimination between the target species on the basis of potential. Moreover, the functional groups of CNFs can hydrogen bond with the analytes’ functional groups (H/O–H) and with the electrode-surface-adsorbed analytes and their aqueous-dissolved species. The rings of CNFs can offer additional π–π interactions to the benzene unit of targeted organic species via solute–sorbent interactions to boost their localized concentration and enhance electrocatalytic redox events. The combined effect of all the above-mentioned interactions gives rise to a sensitive electrochemical scaffold for the discrimination and concurrent biosensing of all the multiple analytes formed due to biochemical reactions in the body such as glucose, uric acid, catechol, H2O2, etc.
MO-based NFs have been synthesized using sacrificial polymers as carriers such as PVP with metal salt, which is then calcined at high temperature to remove PVP while the precursor is oxidized to nanocrystals [51][52]. MOs, by a process called nucleation, grow and align along the direction of the electrospun NFs. The as-obtained NFs have a large surface area with improved optoelectronic properties. Here, the metal/metal oxide NPs exert their electrocatalytic role for biosensing via metal nuclei active sites, and the mechanism follows the conventional heterogeneous catalysis process. Calcination and the concomitant loss of polymer result in the shrinkage in the NF diameter, which causes it to be brittle. The resulting mechanical stress also affects the stability of the sensors, which is improved by blending additives during electrospinning. The process is more complex than producing pristine polymer NFs. Mercapto propyl phosphoric acid (MCPPA)/Cu-doped ZnO NFs prepared from PVA and zinc acetate were immobilized on a glassy carbon electrode (GCE) for the detection of antigen histidine-rich protein 2 (HRP2) for the early and quick detection of malaria with an LOD of 0.6 attogram/mL with high specificity [53]. The HRP2 antigen is released by the lethal malarial parasite into the blood stream of the body. Previous work focused on growing CNFs on glass microballoons with HRP2 as an immunosensor for the detection of a specific antibody via calorimetric or electrical signals [54]. Similarly, Cu-doped ZnO NFs were developed for impedimetrically targeting the same analytes as in [53][54]. Cu was found to improve the sensitivity of the sensor by overcoming the intrinsic resistivity of the ZnO NFs. This woresearkch could be expanded to include other biomarkers of importance. By introducing NFs onto electrodes, the scalability of NF processing is likely, but comes at the cost of developing it in relation to a specific antigen for the detection of malaria. MCPPA doping helps with immobilizing the anti-HRP2 antibodies by introducing the functional groups such as –COO−, etc. The porosity of the sensitive layer offers high-mobility paths and surface activities for target molecules, promoting their interaction and mass transportation across the sensing surface. Cu doping generates an electric field at the interface of the Cu/ZnO heterojunction, along with improved conductivity of NFs. Nano-based immunosensors have been proposed to be used in PoC diagnostic kits. Similarly, a label-free immunosensor for the detection of the breast cancer biomarker, epidermal growth factor receptor 2 (ErbB2), was reported. The PoC device was constructed by using carbon-doped mesoporous ZnO NFs via electrospinning, with fibers ranging in diameter from 50 to 150 nm.
MWCNTs imbedded into crystalline ZnO NFs are covalently bonded to the antibody for the detection of carcinoma antigen-125, which is an ovarian cancer biomarker and can be used for PoC diagnostics due to its high sensitivity and rapid detection. Calcination is optimized to prevent the decomposition of CNTs, and the introduction of –COO− groups helps to bind the antibody to the sensing matrix via the amino groups on the specific antigen via the EDC/NHS coupling reaction. These oxygen functionalities of the fCNTs act as anchoring sites to form hydrogen bonds, which are responsible for the increased adsorption of target analytes [55]. TiO2 NFs have been used to detect malignant cells from patient suffering from colorectal or gastric cancer [56]. Label-free biosensors can detect biomarkers without the need for labeling agents, which can simplify the detection process and reduce the cost of biosensors. Researchers are exploring the use of nanofiber-based biosensors for the label-free detection of biomarkers. An immunosensor based on the wire-in-tube architecture of chitosan modified IrOx NF, where (0 ≤ x ≤ 2) was synthesized by electrospinning for the label-free detection of the α-fetoprotein (AFP) cancer biomarker by controlling the annealing temperature, resulting in a unique morphology with improved surface area. Thus, it was observed that independent nanowires were incorporated into the NF that were separated along the whole length of the NFs. The antibody (Ab) adsorbed on to the highly oriented modified NFs for the amperiometric detection of AFP in human serum with an LOD of 20 pg/mL was confirmed [57].

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