Infectious diseases have emerged as one of the major cause of morbidity, being responsible for several hundred thousands of deaths per year. Currently, most of the diagnostic testing for viral specimens are still examined in a central laboratories and processed in a batch. Nanoscale material-based analytical tools have a significant effect on turning the current analytical methods into diagnostic approaches by transformation their sensing module for the detection of biomolecules such as viruses. Undoubtedly, contemporary biosensing platforms require simultaneous upgrades because of new challenges (technological limitations and biological barriers) in the diagnosis of viral infections due to fast mutation of viruses. Accurate and rapid detection of viruses is vital for rational and effective therapeutic management at early stages of infection, thus avoiding rapid spread of pathogens from person to person. One of the current strategies is combination of nanomaterials with the traditional viral detection techniques. A common approach to detecting the presence of viruses in biological samples is to use inorganic nanoparticles for single-shot analyses, which are usually solution-based assays performed in a cuvette or a microtiter plate and paper-based lateral flow assays (LFAs). Silver and gold nanoparticles functionalized with protein or nucleic acid probes are ideal for assays with colorimetric detection of viral markers due to their plasmonic properties, while another option is to use them for surface-enhanced Raman spectroscopy (SERS) . In addition, fluorescence-based assays can be designed using, for example, nanoparticles doped with lanthanide chelates or semiconductor QDs . Nanomaterial-based biosensors are devices comprising natural biocompatible materials with a convenient platform for detection of pathogenic organisms. These platforms are usually connected to an appropriate data processing system . Growing interest for virus detection by nanomaterial-based biosensors leads to the bioconjugation of CDs to the biosensor structure. As it was already mentioned, fluorescent CDs offer many fascinating properties, such as chemical inertness, high photostability, easy surface modification, low cytotoxicity, and superior biocompatibility [66,67,68]. Thus, CDs have attracted considerable attention among scientists as potential candidates in biosensing. Carbon dots possess adjustable elemental compositions and serve as either electron donors or acceptors. These biosensors, which use immobilized biomolecules such as nucleic acids, antibodies, or enzymes for the detection of analytes, convert a biological response into an electrical or optical signal . Suitable integration of CDs as a main elements of virus genome detection methods appears to enhance the sensitivity and specificity for viral recognition. In this section, recent progress in CD-based biosensors for virus detection and diagnosis is reviewed according to which biosensing approach was used.
The large surface area to volume ratio and plenty of oxygen-rich functional groups in GQDs, as well as their easy functionalization and excellent electrochemical properties, enable applications of GQDs as surface electrode modifiers and signal amplifiers in the evolution of electrochemical biosensors. The principle of these biosensors is the recognition of target analyte sensed by determining the electric response due to the electrochemical reaction of the target analyte with the surface of the modified electrode of the biosensor. As GQDs are considered as biocompatible, the use of GQD-based nanoprobes is facilitated in biomedical diagnostic areas. A very simple, smart, and efficient electrochemical biosensor based on GQDs, which strongly interact with DNA and play a role of an electrode substance, was introduced by Xiang et al. . In a similar approach, a highly sensitive label-free electrochemical platform using a GQD-modified glassy carbon electrode (GCE; most commonly used as a working electrode in electroanalytical chemistry) coupled with a strongly bound specific sequence of DNA molecules as a probe (pDNA) was designed for the detection of potentially life-threatening hepatitis B virus DNA (HBV-DNA). The GQDs were synthesized by a safe and simple pyrolysis process using citric acid . The mechanism of electron transfer from the pDNA-decorated GQD-modified electrode to the electrochemically active species K3[Fe(CN)6] was monitored by differential pulse voltammetry (DPV). The low values of peak current caused by the presence of pDNA on the surface of the GQD-modified electrode increased due to the elimination of electrostatic repulsion that occurs after the addition of HBV-DNA, where HBV-DNA-pDNA duplexes were released in a concentration-dependent manner. This strategy demonstrated an excellent linear detection range from 10 to 500 nM and provided a detection limit of only 1 nM, the best value among carbon-based electrochemical DNA biosensors. In addition, it should be noted that this fluorophore-free labeling approach also represents an enzyme-free signal amplification strategy in contrast to standard detection assays. Another advantage is that the biosensor prepared in this way is not only safe and highly sensitive, but also inexpensive. This electrochemical platform for HBV-DNA detection and DPV results are illustrated in Fig. 4.
The use of antigens and their combination with specific antibodies as an efficient strategy has opened new doors for designing of improved assay for hepatitis C infection diagnosis. Compared to conventional immunoassays such as ELISA, which can require several days, electrochemical analyses are much faster. At the same time, key parameters such as signal amplification, sensitivity, and stability must be ensured for accurate sensing. For example, Valipour and Roushani  fabricated a label-free electrochemical immunosensor based on a functionalized nanocomposite of silver nanoparticles (AgNPs) decorated with thiolated GQDs (GQD-SH) as GCE modifier for biorecognition of the hepatitis C virus core antigen (HCV). Due to the small size of GQDs, which is less than 100 nm, the resulting signal is amplified and the large surface area allows immobilization of a large number of antibodies on the surface of the nanocomposite. To remove the redox reaction products, the GCE was polished with alumina powder and thoroughly rinsed with deionized water. Subsequently, the electrode was soaked in the GQD-SH solution, followed by the immobilization of AgNPs on the surface of the GQD-SH/GCE via Ag–S bond formation. In the next step, anti-HCV antibody was covalently attached to the surface of AgNPs via its terminal amino group (−NH2). In order to experimentally determine the immunorecognition reaction, a biological molecule of riboflavin was used as a redox probe for the detection of HCV using differential pulsed voltammetry measuring. Along with the specific immune recognition, the decrease in the oxidation signal response of riboflavin was investigated. The proposed device showed a wide linear concentration range (0.05 pg mL−1 to 60 ng mL−1) with the detection limit of 3 fg mL−1. Finally, the application of this platform was evaluated also in clinical diagnosis where HCV core antigen was reliably detected in real samples of spiked human serum from patients. This electrochemical device for hepatitis C virus detection is illustrated in Fig. 5. The main feature of GQD-SH in this new type of electrochemical immunosensor is their high surface-to-volume ratio, biocompatibility, dispersibility in relevant solvents, and unique electronic structure that leads to sensitive detection of human serum samples with enhanced signal.
Another sandwich-type electrochemical immunosensor utilizing GQDs was developed for the detection of an oncogenic retrovirus, avian leukosis virus subgroup J (ALVs-J), using apoferritin-encapsulated Cu (Cu-apoferritin) nanoparticles for enhancing of the signal amplitude . GQDs were chemically synthesized via cleaving graphene oxide with the assistance of microwave irradiation . The immunosensor setup involved the Fe3O4@GQD hybrid platform for the immobilization of secondary ALVs-J antibody (Ab2) and Cu-apoferritin nanoparticles as electroactive probe (Fe3O4@GQD/Ab2–Cu-apoferritin). Again, the huge surface area of GQDs prepared by the well-known Hummers’ method enabled immobilization of large number of antibodies on the surface of the immunosensor, resulting in a significant increase in signal. Compared to larger carbon nanotubes, these small particles of 3 to 20 nm exhibit a greater number of carboxyl groups and better conductivity. In addition, their excellent electrocatalytic effect has been observed. In this study, a very nice comparison of the signal amplification abilities of GQDs and apoferritin was investigated. The immunosensor with GQDs showed a larger current shift (ΔI 1/4 19.96 μA) than the immunosensor without GQDs. This was mainly attributed to the larger free surface area that GQDs provided for the conjugation of Cu-apoferritin and Ab2. More significantly, the electrochemical immunosensor with apoferritin exhibited a higher peak current (ΔI 1/4 47.74 μA) than the immunosensor without apoferritin. These results confirmed that the use of apoferritin can accommodate more electroactive probes and GQDs can immobilize more Cu-apoferritin and Ab2. Therefore, the use of GQDs and apoferritin significantly amplified the immunosensor signal. Deposition of GQDs onto the bare GCE was done using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as a cross-linker and by subsequent incubation in a primary ALVs-J antibody (Ab1) solution. The quantification of the target ALVs-J virus was realized when ALVs-J was captured onto the Ab1-immobilized electrode surface, followed by a specific immunorecognition reaction with the Ab2 antibody on the hybrid surface with the subsequent formation of the sandwich structure. Electrochemical behavior was evaluated after each step of the surface modification and biosensing studies were conducted by cyclic voltammetry (CV) and DPV, respectively. This electrochemical immunosensor was able to detect as low as 115 TCID50 mL−1 with a wide detection range from 102.08 to 104.50 TCID50 mL−1.
New developments in nanotechnology pave the way to robust electrochemical detection of viruses, especially using impedimetric biosensing. Impedimetric-based immunosensors enable to measure the changes in charge conductance and capacitance according to varying concentration in the target analyte immobilized on the immunosensor surface . Applying GQDs as a high-performance material displays promising features for constructing impedimetric biosensors. Moreover, doping of GQDs with chemically bonded nitrogen and sulfur atoms allows to tune their electrochemical properties as well as increasing the amount of the anchoring sites for the adsorption of metal ions. Chowdhury et al.  recently reported a discovery of an electrical pulse-induced immunosensor based on the use of nitrogen and sulfur co-doped graphene GQDs (N,S-GQDs) and gold-embedded polyaniline nanowires (AuNP-PAni) for quantifying the hepatitis E virus (HEV) by an impedimetric response. Hepatitis E virus (HEV) infection is known primarily as a cause of acute hepatitis, a viral disease in which even low levels of the virus pose a potentially fatal threat. To fabricate a biosensor electrode, a N,S-GQD@AuNP-PAni nanocomposite—covalently conjugated by the specific anti-HEV antibody—was used, followed by an interfacial self-oxidation-reduction polymerization on a clean GCE. The AuNP-PAni served as enhancers of the electron transfer process and also provided high surface area for the target HEV. The presence of N,S-GQDs on the electrode surface played an important additive role in keeping high conductivity in impedimetric response.
As already mentioned for N,S-GQDs, the chemically bonded nitrogen atom can significantly improve the electrochemical properties by changing the electronic characteristics, and sulfur can increase the number of anchor sites for adsorption on noble metal nanoparticles. N,S-GQDs are also used to enhance the electrochemical activity and conjugation of antibodies through their edge carboxyl groups. By combining the three components, PAni, AuNPs, and N,S-GQDs, a nanocomposite was constructed that has a synergistic effect of its organic and inorganic parts. Therefore, the nanocomposites exhibit excellent electroactivity in analyte solution. To achieve more sensitive detection of HEV, the biosensor was exposed to different external pulses at the time of the virus loading. Such an electrochemical impedance spectroscopy (EIS) biosensing platform could efficiently determine and discriminate various HEV genotypes (G1, G3, G7, and ferret HEV) in wide concentration range from 1 fg mL−1 to 100 pg mL−1 in cell culture supernatants with a detection limit of 0.8 fg mL−1. To confirm the applicability of the method developed in this study, fecal samples of a HEV-infected monkeys were also examined with sensitivity comparable to that achieved by a reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR).
Nowadays, electrochemical biosensors are the most commonly used for detection of various viruses. In spite of the advantages such as low cost, simple instrumentation, and rapid response, this method is sensitive to sample matrix effects, has small temperature range, and short instrument life. Thus, further optimization of this method is proposed.
In the recent past, the range of protein-based virus identification methods was extended by so-called aptamer-based biosensors, also called aptasensors, which consists of a selective and specific aptamer probe displaying robust binding affinity towards the proteinaceous part of viral materials . Aptamers are similar to antibodies, but possess more advantages including greater stability, a smaller size, low cost, and an easier synthesis process. The electrochemical approach which utilizes the aptamer as biorecognition element was introduced, for instance, Ghanbari et al. . They have developed a label-free technique, taking GQD nanocomposite as the electrochemical aptasensor probe for the ultrasensitive quantification of a core antigen from the HCV. To fabricate the aptasensor, GQDs served as an appropriate immobilization substrate for aptamers through π stacking interactions, thus enhancing the aptamer molecule accumulation on the GCE surface. The surface composition of such an aptasensor was monitored by EIS, as well as measuring by CV and DPV techniques after each functionalization step. In this report, the EIS biosensor was able to efficiently determine the target HCV core antigen, which exhibited a wide linear concentration range from 10 to 400 pg mL−1 with the detection limit of 3.3 pg mL−1. This biosensor probe can efficiently determine the target analyte even in human serum samples.
Among all kinds of biosensor platforms, paper-based biosensors are commercially attractive alternative due to their easy preparation, handling, availability, transportability combined with low-cost and effective manufacture, thus surpassing the screen printed glassy carbon-based electrodes [79, 80]. Lateral flow biosensors do not require sophisticated instrumentation and enable very easy handling. Although a lot of lateral flow test strips have been reported, their sensitivity limits their subsequent application. A sandwich type bioimmunoassay architecture, which is utilized as a unique research tool for the highly biospecific recognition interaction between an antibody and antigen for fast and sensitive key-lock detection, can be also integrated with a lateral flow test strips. Recently, considerable effort has been put in developing a test strip system for the detection of the virus by choosing various labels as a signal reporter such as gold nanoparticles (AuNPs) , colored latex beads , and quantum dots . Moreover, these test strips can be also coupled with fluorescent CDs as tags to further improve the detection sensitivity. For instance, Xu et al.  proposed a point-of-care immunoassay biosensor of fluorescent CD/SiO2 nanospheres (CSNs) for the detection of sever fever with thrombocytopenia syndrome virus (SFTSV) with high ultra-sensitivity. The CSNs prepared by a simple co-hydrolysis of silanized CDs with tetraethyl orthosilicate (TEOS) used as a template were coupled with lateral flow test strips. Before the immobilization of the antibody capturing the SFTS virus, the carboxyl-terminated groups of CD/SiO2 were activated by using EDC as a coupling agent and NHS as an activator. Due to the excellent fluorescent properties of CSNs, the biosensor provided the benefit of having a longer assay lifetime and good selectivity. Based on this principal, the content of the SFTS virus in buffer was successfully visually observed with a limit of detection 10 pg mL−1. Subsequently, four different antigens (HCG, AFP, CEA, and CA125) with a concentration of 1 μg mL−1 and the blank sample of phosphate buffer were immobilized individually in five test strips and were used for the examination of the method selectivity. There was no observable any fluorescent intensity of the test line for those nonspecific protein samples, whereas the SFTSV monoclonal antibody specifically recognized the corresponding antigen. Finally, the application of this method was evaluated also in clinical diagnosis where SFTSV was reliably detected in real samples of a patient’s serum. This type of capture assay for SFTS virus is illustrated in Fig. 6.
Later on, in 2021, the same group—Xu et al. —also extended the utilization of the immunofluorescent CD/SiO2-based lateral flow strip platforms to sensitive, rapid, and specific detection of Zika virus. The traditional method of Zika virus detection involves amplification of the virus genome, which is time-consuming and requires well-trained technicians. The lateral flow test has been successfully used to detect viruses, cancer, small molecules, bacteria, etc. because of its advantages of immediacy, simplicity, ease of use, and ease of interpretation. Fluorescent CD/SiO2 (FCS) structures with various contents of silanized CDs were doped onto the inner surface of tree-like shapes SiO2 colloids as the template host via strong Si-O bonding formation. Then, the ZK01 antibody was conjugated with FCS spheres through its carboxyl groups. The immunorecognition reaction was monitored by observing the test results under a 365-nm ultraviolet lamp. The immunosensor showed a wide linear concentration range (10 pg mL−1 to 1 μg mL−1) with the visual detection limit of 10 pg mL−1 and was applied to the analysis of spiked human serum. The measurement of the Zika NS1 protein in a simulated human serum revealed a promising diagnostic applicability. Compared with a traditional AuNP-based lateral flow assay, the results of this FCS-analysis showed 100× higher sensitivity. In addition, Xu et al. used a similar system composed of dendritic silica nanospheres coated with red emissive CDs to detect SARS-CoV-2 nucleocapsid proteins with a sensitivity of 10 pg mL−1 and a linear response up to 1 μg mL−1 . Moreover, this system can be used in the future as a new and cost-effective simple detection method, especially in poor regions of the world.
To the best of our knowledge, only one study has recently reported the use of GQD-based photoelectrochemical (PEC) nanobiosensors as a promising low-cost approach for virus detection. When the photoelectrochemical biosensing strategy is adopted, the biological interactions between the analyte and the biorecognition element result in the photoelectric conversion process under light illumination and applied potential where the photocurrent is recorded as the detection signal. Photoelectrochemistry appears to be a challenging research topic because of its remarkably high sensitivity and low background signal, along with being particularly appropriate for biomolecule determination at very low concentrations [87,88,89]. Moreover, the PEC system possesses specific features such as rapid analytical response, simple instrumentation, and easy miniaturization. The principal of the PEC biosensors consists in the generation of a photocurrent signal during the biological interaction between bioreceptor and the target analyte . This PEC reaction is accompanied with the energy transfer and charge transfer processes under light illumination between the photoactive element and electron donating/accepting moieties . With the progress of nanotechnology, CDs have been used as photoactive species for fabricating PEC biosensors . Again, the large specific surface area and good biocompatibility of CDs provide an opportunity to attach and load large amount of biomolecules and help to maintain their bioactivity significantly. Nevertheless, research in this area is in its infancy, and, thus, the photoelectrochemical mechanism is required to be further examined. In 2018, Ahmed and his team  constructed an optoelectronic immunosensor for fowl adenoviruses (FAdVs) identification by exploring the activity of GQDs assembled on a gold nanobundle (Au NB) film. In this work, GQDs were synthesized with the assistance of a benchtop design autoclave. This approach utilized a modified layer-by-layer assembly technology as a promising strategy to the deposition of an Au NB film on a carbon printed electrode through the use of L(+) ascorbic acid, gold chloroauric acid, and poly- L -lysine (PLL). A nanohybrid structure consisting of GQDs and Au NBs was coupled with anti-adenovirus antibody via an electrostatic bonds through positively charged PLL and via an amide bonds through cysteamine, respectively, as depicted in Fig. 7. A local electric signal enhancement under UV light irradiation, where Au NBs and GQDs came close to each other, was directly co-related with the FAdV antigen concentration in the detection ability up to 10 plaque-forming unit (pfu) mL−1 and 50 pfu mL−1 with the limit of detection (LOD) value of about 8.8 pfu mL−1 and 37.2 pfu mL−1 in buffer and chicken blood media, respectively. Compared with a traditional gold enzyme-linked immunosorbent assay (ELISA) methods, this optoelectronic biosensing assay provided the excellent sensitivity (100 times higher).
Last but not least is the detection approach using the Golgi apparatus, which was introduced by Li et al. . The method combined the benefits of L-cysteine-functionalized carbon quantum dots (5–10 nm) and silica nanoparticles (40–80 nm), and has been successfully applied for targeting and monitoring the morphological changes of the Golgi apparatus during the process of a viral infection with respiratory syncytial virus under visualization by optical and electron microscope. After the infection, the Golgi broke down into small fragments and become scattered. Here, thanks to the stereo configuration and free thiol groups of L-cysteines on their surface, fluorescent carbon quantum dots can target the Golgi with high specificity. Moreover, due to the extraordinary photostability and biocompatibility of these CDs, the system is appropriate for long-term in situ imaging of the Golgi. The authors thus developed a promising approach to the imaging strategy for early diagnosis, drug delivery, and subsequent therapy of Golgi diseases.
Surprisingly, less than 20 papers have been published so far in which CDs have been used to construct biosensors for virus detection (see Table 1). In our opinion, there is still a great opportunity to push forward the effort to develop new breakthrough biosensors based on emerging CDs and their unique optical properties towards fast, sensitive, and specific sensors that could be easily constructed in a low-cost process. Why not have paper strips or antigen assays containing suitably modified CDs where only the very simple “light-on/light-off” detection principle is used to detect, for example, Covid-19?