Viruses are infectious entities, in which one kind of nucleic acid, either DNA or RNA, is wrapped in a protein shell with a diameter ranging from 16 nm to over 300 nm^[1]. The nucleic acids are sometimes also encapsulated in a lipid membrane in some specific species. Since viruses do not contain ribosomes or any other cellular organelles, their only means of reproduction is to infect the living cells and replicate by using the ribosomes of host cells^[2]. Emerging and reemerging viral infections can cause severe respiratory tract, intestinal tract, skin, single organ, multi-organ, nervous system, and hematological diseases, and adversely affect human the health^[3-6]. In addition, numerous outbreaks of large-scale infection have occurred worldwide, especially the current outbreak of novel coronavirus disease 2019 (COVID-19)^[7]. Due to the severity and damage caused by such infections, rapid, accurate, available, user-friendly, and low-cost detection methods for the detection of viruses are necessary^[8,9]. The existing methods, such as virus culture, polymerase chain reaction (PCR)^[10], enzyme linked immunosorbent assay (ELISA)^[11] and other serological methods^[12], are widely used in clinical practice. These methods are extremely sensitive and specific, however, they require expensive equipment and professional operators and suffer from major drawbacks of being time consuming and labor intensive^[13,14].

Nanobiosensor is a kind of novel biosensor, which primarily depends on the specific recognition and interaction of nanomaterials with the molecular substances, such as enzymes, antigens, antibodies, and DNA, to transduce as optical and electrochemical signals^[15]. Moreover, due to the high signals and large surface volume ratios of nanomaterials, nanobiosensors can determine extremely low analyte concentrations in a variety of biological samples with high accuracy, sensitivity, and specificity^[9,16]. Interestingly, various kinds of nanomaterials, such as gold nanoparticles (Au NPs)^[17], carbon nanotubes (CNTs)^[18], magnetic nanoparticles^[19] and quantum dots (QDs)^[20] can be used for nanobiosensors. Among them, QDs are one of the best characterized nanomaterials with versatile optical and electrical properties, which can function as semiconductor nanocrystals and mainly made up of group II–VI, II–V or IV–VI^[21]. Compared with organic fluorescent dyes, the QDs have unique properties such as broad excitation range, symmetric photoluminescence, multiplex staining, long fluorescent lifetime, and high resistance to photobleaching^[22,23]. The main synthesis methods for QDs include organometallic synthesis and aqueous synthesis, and the aqueous QDs have been found to be more suitable for bioapplication^[24]. In addition, the biofunctionalization of QDs is necessary, which can attach different biomolecules (oligonucleotides, proteins, peptides, and small molecules) onto the surface of QDs by various interactions (electrostatic interaction, covalently coupling, and metal-ligand interaction).

Recently, QDs have been actively exploited for virus detection by integrating novel analytical methods (e.g., fluorescence resonance energy transfer, aggregation induced quenching, magnetic separation, and enzyme-linked immunosorbent assay) with the functional materials (e.g., gold nanoparticle, graphene oxide, magnetic bead, and SiO₂ microsphere)^[25-28]. However, there are limited reports that have extensively reviewed strategies for QDs-based virus detection. Herein, we review the recent advances in QDs-based biosensors for viral nucleic acid, antibody, and antigen detection (Fig. 1). First, the synthesis and biofunctionalization of QDs was described. Then, we have mainly focused on viral nucleic acid detection, including nucleic acid hybridization, nucleic acid amplification, and probe signal enhancement. Later, we have introduced the strategies for viral antibody and antigen detection, namely, QDs-based immunoassays, including heterogeneous immunoassays (enzyme-linked immunosorbent assay, magnetic bead-based immunoassay, and lateral-flow immunochromatographic assay) and homogeneous immunoassays (fluorescence resonance energy transfer-based immunoassays). Finally, we have discussed the various challenges and future direction of QDs-based virus detection.

QDs are typical zero-dimensional nanomaterials, including II–VI, III–V, or IV–VI nanomaterials^[29]. However, compared with the traditional fluorescence dye and fluorescence proteins, QDs exhibit excellent optical properties, such as broad absorption spectrum, narrow and adjustable emission, high quantum yield, good photostability, and easy biological functionalization^[30,31]. The methods used for synthesis of QDs include organometallic synthesis^[32] and aqueous synthesis^[33]. The QDs obtained by organometallic synthesis have uniform particle size, high quantum yield and good stability, but have been found to be highly toxic. The aqueous synthesis method is simple, green, and easy to surface functionalization, which can be directly used in various biological systems. The biological functionalization of QDs involves use of proteins, nucleic acids, or small biomolecules to modify the surface of nanoparticles, which has a wide range of applications in biosensors and bioimaging^[34]. The major methods include electrostatic interaction, covalently coupling, and metal-ligand interaction.

Organometallic synthesis is conducted to obtain QDs by using hot injection or heating-up methods to facilitate the nucleation and growth of the precursors in organic solvents^[32]. The hot injection method involves rapid addition of the precursor to the hot organic solvent for nucleation, and the precursor is continuously added to promote the growth^[35]. The heating-up method is used to make the precursor locally supersaturated to nucleate and grow significantly by heating^[36]. Water-soluble modification is necessary for the bio-application of organometallic QDs, which includes the ligand exchange^[37], hydrophobic coating^[38], and silanation^[39]. Ligand exchange refers to one end of the hydrophilic molecule which contains sulfhydryl, pyridine, imidazole, or other structures, can replace the original ligand on the surface of QDs, and the hydrophilic groups at the other end to maintains its solubility in water^[40]. The dissociation and binding of the ligands is a dynamic and reversible process, which can easily lead to the aggregation of QDs and the reduction of fluorescence intensity. Hydrophobic coating indicates that QD is coated by amphiphilic polymer, such as polymaleic anhydride, polyacrylamide, or polyethylene glycol, and the hydrophobic chain is embedded in the hydrophobic ligand on QD surface, with the hydrophilic end facing outward to achieve water solubility^[38]. The method has good photostability, but the size of particles after coating is relatively large. Silanization can form the silica shell on the QD surface to cause it to stably disperse in water. Silica is non-toxic substance, has good biocompatibility and can significantly inhibit QDs photodegradation^[41]. Silanization uses non-toxic silica, which has good biocompatibility, but has not been widely used due to its complex operation.

The aqueous synthesis of QDs mainly includes microwave^[42], reflux^[43], and hydrothermal methods^[33], among which hydrothermal method is the most widely used. In the hydrothermal method, thiol-phosphorus precursors and metal precursors are prepared, respectively, and then mixed it to form QDs crystal nuclei, which are then transferred into Teflon-lined stainless-steel autoclave to further grow into QDs^[44,45]. Thiol-phosphorus precursor is usually obtained by the reaction of Te/Se and NaBH₄ under anaerobic conditions, and its product can be easily oxidized, so the transfer process requires rapid and skilled operation. Zhanget al. developed a one-pot hydrothermal method to prepare QDs using Na₂TeO₃ as Te source to simplify the process (Fig. 2(a))^[46]. In this method, Te²⁻ was obtained by reacting Na₂TeO₃ with NaBH₄, which could directly interact with the metal precursor and did not involve the transfer of Te precursor. Surface modification of QDs can affect the particle size, shape, luminescence efficiency, cytotoxicity, cell penetration, and non-specific adsorption of QDs^[47]. Thiols are the most commonly used surface modified ligands, which can interact strongly with the metal ions and stabilize QDs. QDs modified by monomercaptan ligands can exhibit good luminescence efficiency, but poor photostability, easy aggregation, and narrow pH range. Multimercaptan ligands have stronger interaction with the metal ions and can be used to modify QDs to obtain better stability. Maoet al. developed innovative methods for preparing the stable aqueous QDs using dithiol as the surface ligand, which could markedly improve fluorescence quantum yield and stability^[48-52]. Zhanet al. used amino group and tetramercaptan as potential ligands for QDs synthesis, which can stably exist in high concentration of the salt, cell culture medium, or glutathione solution, and possess stable fluorescence properties in the pH range of 2–14 (Fig. 2(b))^[53]. In addition, other ligands, such as imidazole and adenosine, can also stabilize QDs, but due to their poor interactions with metal ions, co-stabilizers are required. Wanget al. designed and synthesized a ligand containing both imidazole and dithiol, which exhibited the advantages of strong interaction between dithiol and metal ions and anti-oxidation of imidazole (Fig. 2(c))^[37].

The combination of biomolecules and QDs via electrostatic adsorption is one of the simplest methods employed for biofunctionalization of QDs. Bhanget al. prepared HA-QDs by using negatively charged hyaluronic acid (HA) and positively charged QDs to detect hyaluronidase (Fig. 3(a))^[54]. Although electrostatic adsorption is a simple and effective technique, it is difficult to avoid non-specific adsorption. Moreover, some biomolecules, such as protamine and sodium heparin, could easily cause aggregation and self-quenching of QDs.

Covalent coupling is the most common method employed for biofunctionalization of QDs, mainly including the reaction of amino and carboxyl, hydrazine, and aldehyde, phenylboric acid and diol, and maleimide and sulfhydryl group^[55-57]. Carbonized diimide chemical method has been used to activate the carboxyl group to obtain amide and promote the reaction between amino and amide to form ester^[58]. Nie and Chanet al. covalently coupled thioglycolic acid stabilized CdSe/ZnS QDs with transferrin by using carbonized diimide chemical method to obtain transferrin-QDs^[59,60]. Transferrin-QDs can specifically recognize the different surface receptors and can effectively enter into cervical cancer (HeLa) cells, which were used for cell imaging for the first time. Generally, antibodies contain the sulfhydryl groups, which can often react with maleimide modified QDs to quickly facilitate antibody labeling^[55]. Multicolor QDs can be used to label the different antibodies to detect multiple antigens^[55]. Although the conjugation between QDs and biomolecules is very stable, these coupling reactions can easily lead to the reduction of luminescence efficiency and stability of QDs. Recently, a novel covalent coupling method, click reaction, has attracted significant attention due to its mild reaction conditions, high coupling efficiency, and minimal cross coupling tendency. Click reaction usually requires copper ions as a catalyst, but it can effectively quench the QDs fluorescence and has cytotoxicity^[61]. Maoet al. developed azide-DNA functionalized QDs for GOx labeling by copper-free catalytic click reaction to produce GOx-QDs, which could be employed for the rapid blood glucose detection with high sensitivity (Fig. 3(b))^[62].

Inspired by the biomineralization reaction between the metal ions and histidine-rich proteins, Clappet al. modified CdSe/ZnS QDs with histidine-containing biomolecules, which showed strong binding force and fast binding speed^[63]. Because histidine is easy to fuse with different proteins, it is suitable for establishing the connection between QDs and proteins. Metal ions also have strong binding capacity with sulfhydryl or imidazole and are commonly used in biofunctionalized QDs^[64]. In another study, Wu et al. synthesized a sulfhydryl modified DNA and prepared a two-dimensional QD-beacon through the combination of sulfhydryl and metal ions (Fig. 3(c))^[65]. The BHQ3 and chlorin e6 were modified in DNA, which were used for imaging and photodynamic therapy, respectively. However, without directly targeting the microRNA, QDs fluorescence was quenched by BHQ3 and the photodynamic therapy was inhibited. In the present of the target microRNA, QDs fluorescence was recovered and the photodynamic therapy was inactivated. Maet al. designed a BHQ and phosphorothioate co-modified DNA to prepare QD nano-beacons with controllable valence for single RNA imaging and virus dynamic tracking in the live cells^[66].

Due to high quantum yield, photobleaching resistance, and high signal-to-noise ratio, QDs have displayed good application potential for the early diagnosis of viruses and the study of infection mechanisms in recent years. A number of QD-based methods have been developed for viral gene detection and protein immunoassay. The genetic material consisting of the nucleic acid plays a significant role in the virus replication, proliferation, heredity, and evolution, and is the main biomarker for the diagnosis of virus infection^[67]. The principle of nucleic acid detection is based on nucleic acid hybridization with high specificity. A variety of DNA functionalized QDs probes have been constructed for viral nucleic acid detection, and the detection sensitivity was further improved through nucleic acid amplification and signal enhancement^[22,68-70]. Immunoassay depends primarily on the specificity of antibodies and antigens binding to achieve the diagnosis of virus infection. However, compared with nucleic acid detection, the immunoassay is simple and convenient, does not require expensive instruments as well as the technical personnel, and is suitable for rapid virus screening. Immunoassays can be mainly divided into homogeneous and heterogeneous assays. Heterogeneous system can remove the free components from target analyte through washing step, which is an immensely popular analytical method with high sensitivity and versatility. On the contrary, homogeneous system has no solid support and does not require washing and separation process. It is suitable for the rapid detection and can improve sensitivity through signal amplification.

The most common method for nucleic acid detection is to recover the fluorescence signals through promoting the hybridization of target nucleic acid and DNA probes, such as molecular beacon and DNA functionalized QD. QD hybridization probe is constructed by conjugation of the quencher with QD, and four conjugation methods have been identified, including electrostatic interaction, π–π stacking interaction, covalently coupling, and metal-ligand interaction. Jianget al. prepared positively charged carbon QDs and combined them with Dabcyl-modified DNA through electrostatic interaction to obtain QDs hybridization probes for facilitating extremely sensitive detection of the human papillomavirus 16 nucleic acid (Fig. 4(a))^[71]. The coupling of carbon QDs and TARMA-modified DNA resulted in fluorescence quenching, and complementary hybridization between the target nucleic acid and DNA probe induced the fluorescence recovery. Because of the strong quenching efficiency of TARMA by carbon QDs, this method was found to display high sensitivity. To further improve the sensitivity, Tianet al. enabled fluorescence resonance energy transfer (FRET) from DNA functionalized QDs to carbon nanotube (CNT) through π–π stacking interaction (Fig. 4(b))^[18]. CNT was considered as an effective receptor in FRET system due to its strong absorption capacity, thereby resulting in high signal-to-noise ratio. After hybridizing with H5N1 nucleic acid, the generation of the rigid DNA double strand blocked the FRET effect and caused fluorescence recovery.

The specificity and reproducibility of the nucleic acid detection need to be enhanced due to the relatively weak binding of electrostatic and π–π interaction. Nanobeacon is prepared by covalent coupling, which can enhance the binding of QD and quencher for the specific and reproducible detection of viral nucleic acid. The commonly used quenchers include Cy5^[72], black hole quencher (BHQ)^[73], and Au NPs^[74]. Adegokeet al. assembled the BHQ-DNA modified QDs on the surface of Au-NPs, and the fluorescence of QDs was quenched by BHQ without targeting the nucleic acid (Fig. 4(c))^[75]. After hybridization with Zika RNA, QD fluorescence was recovered and further enhanced under the effect of the surface plasmon resonance with Au NPs, with the detection limit being as low as 1.7 copies/mL. In addition, to avoid the influence of covalent coupling on the properties of QDs, our group has constructed BHQ-DNA functionalized QDs through the interaction of Cd²⁺ and phosphorothioate in BHQ-DNA (Fig. 4(d))^[66]. This method did not affect the fluorescence of QDs, and the amount of DNA modified on QDs can be precisely controlled by the number of phosphorothioate groups. The developed QD nanobeacon was found to be suitable for imaging a single HIV-1 RNA in the latent infected cells, thus providing a platform for the analysis of single copy nucleic acid in the live cells.

Nucleic acid amplification is a method that can amplify the nucleic acid to be evaluated through the catalyzation of the nuclease or nucleic acid self-assembly and then can aid in its detection. Nucleic acid amplification has been divided into variable temperature amplification and isothermal amplification according to the temperature control. Polymerase chain reaction (PCR) is considered as the most common variable temperature amplification method because of its high amplification efficiency^[76]. For instance, Chenet al. used biotin-primer and digoxin-primer/TAMRA-primer to amplify SARS-CoV-2 N gene /influenza B virus by PCR, and the generated amplicon served as a potential bridge between the digoxin antibody/TAMRA antibody modified test strip and streptavidin-QDs (SA-QDs) to facilitate the fluorescence detection of SARS-CoV-2/influenza virus (Fig. 5(a))^[77]. However, compared with PCR, isothermal amplification possesses the advantages of rapidity, high efficiency, good specificity and lack of requirement of variable temperature instrument. Loop-mediated isothermal amplification (LAMP) has received considerable attention in nucleic acid detection due to its simple temperature control technique and short detection time^[78]. It is characterized by designing four distinct primers for six regions on the target gene, by using DNA polymerase with chain replacement activity to amplify the nucleic acid under constant temperature condition, which can achieve about 10⁹ times amplification in 15–60 min^[79]. Leeet al. amplified the nucleic acid of SARS-CoV-2 by LAMP, which resulted in the negatively charged polyphosphate (Mg₂P₂O₇), and then aggregated with positively charged cysteamine modified CdSeS/ZnS QDs lead to fluorescence quenching of QDs (Fig. 5(b))^[80]. The fluorescence decrease observed in QDs could be used for SARS-CoV-2 detection.

In addition, enzyme-free amplification techniques have been also widely used in nucleic acid detection. Catalyzed hairpin assembly (CHA) is achieved by the target-induced DNA hairpin self-assembly and has been proven to be an effective enzyme-free amplification technique^[81]. Liet al. described a novel colorimetric determination of hepatitis C virus (HCV) RNA based on the magnetic separation and catalytic hairpin assembly (CHA) (Fig. 5(c))^[82]. Hairpin 1 (H1) was modified onto MBs and opened by the target HCV RNA, which further unfolded the biotin-hairpin 2 (H2) to release the target HCV RNA for further hybridization. Glucose oxidase modified with SA was bound onto biotin-H2, which can catalyze the glucose to produce H₂O₂ to induce in situ degradation of the silver nanocomposite in graphene QDs silver nanocomposites (GQD/Ag NCs). GQDs fluorescence recovered after interaction of H₂O₂ and silver nanocomposite was used for HCV RNA detection. This method is simple, stable, and sensitive with detection limit of 24.8 pM, which can be effectively used for the rapid screening of virus infection.

In addition to amplifying the various targets present in sample, the reporter signal enhancing is another effective method for augmenting sensitivity. Assembly of QDs can effectively improve the reporter signals, such as liposome coating QDs and QDs layer-by-layer assembly. Zhouet al. described a novel approach for the sensitive detection of low-abundance HIV DNA by using liposome coating QDs (Fig. 6(a))^[83]. Liposomes were used to encapsulate the QDs and then modified with DNA to form the DNA-L/QD complex. Thereafter, the target HIV DNA can link the DNA-L/QD complex and DNA modified magnetic beads (MBs). which can be detected with single-particle detection techniques. This method allows for the optimal detection of HIV DNA at attomolar concentration without any target amplification, which can reach up to 3 orders of magnitude in comparison with that of single QDs-based method. Cui et al. developed a signal enhancement method for the detection of HIV DNA via a QD layer-by-layer assembled polystyrene microspheres (PS) to simplify the assembly process (Fig. 6(b))^[84]. PS was used as the core and was able to attract the biotin-QDs by electrostatic interaction, and then could react with SA-QDs to produce layer-by-layer QDs assembly on the surface of the PS (PS-QDs). In the presence of the target HIV DNA, biotin-DNA conjugated with the PS-QDs as the detection reporter was bound onto 96-well plate immobilized with captured probes to produce the fluorescence signals, which could reach ~10 amol of target detection. This method has been found to be simple and controllable.

Amplification based on the nuclease, such as duplex specific nuclease (DSN) and clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nuclease, could enhance the detection sensitivity and simplify the process^[85-87]. For instance, Wang et al. developed one-step method for enterovirus 71 (EV71) infection-related miRNA based on DSN-assisted target amplification and magnetic separation (Fig. 6(c))^[88]. DNA functionalized QDs were covalently coupled onto the magnetic beads, which could be effectively released by the target hybridization and DSN-assisted DNA cleavage. After the magnetic separation, the released QDs were used for EV71 infection-related miRNA concentration determination. A novel signal amplification method was designed for virus detection based on CRISPR system. However, due to the recognition and cleavage of the target nucleic acid, CRISPR and CRISPR-Cas systems, discovered as adaptive immune systems for bacteria against invading nucleic acids, have gained significant attention in biosensor^[89]. For example, Baoet al. developed a novel method for the detection of African swine fever virus by combining CRISPR-Cas mediated nucleic acid probe cleavage and highly sensitive reporter of QDs (Fig. 6(d))^[90]. In the present of target, biotin ssDNA probe could be cleaved by activated Cas12a, but SA-QDs were unable to conjugate with MB, which can be detected by portable instrument after the magnetic separation. This method has displayed high sensitivity and could be used for the visual detection coupled with easy operation. Combining CRISPR-based signal amplification and QD assembly-based signal enhancement could further improve the sensitivity. Zhanget al. proposed another method for SARS-CoV-2 detection which used the combination of isothermal amplification-CRISPR/Cas13a with QDs microspheres (Fig. 6(e))^[91]. SARS-CoV-2 Spike gene was amplified by reverse-transcription recombinase-aided amplification and transcribed into RNA to specifically activate the cleavage of Cas13a. After Cas13a activated cleavage of biotin-RNA-FAM, QDs microspheres were not able to bind and hence not detected on the test strip. The SARS-CoV-2 Spike gene could be detected according to the QDs signals on strip, which is fast, sensitive, and specific with no need for complicated instruments. These advantages demonstrate its potential application for in situ screening of SARS-CoV-2 during pandemic, especially in the remote and underdeveloped areas with limited equipment and resources.

In heterogeneous detection system, the antigen-antibody complex is physically separated from the free components. According to the separation interface, it has been primarily divided into enzyme-linked immunosorbent assay (ELISA), magnetic bead-based immunoassay (MBIA), and lateral-flow immunochromatographic assay (LFIA). Among them, ELISA can combine the specific antigen-antibody recognition and facilitate efficient biocatalysis of enzymes^[92]. The output signals are generated by catalyzing the substrate through the enzyme coupled with an antibody or antigen. Because of its high throughput, low cost, and straightforward readout, it is the most widely used immunoassay. The sensitivity of ELISA system based on the colorimetric signal is too low to detect low-level proteins, while fluorescence signal can significantly increase the sensitivity by 1–2 orders of magnitude^[93], and QD is considered as a suitable candidate for fluorescence reporter. Wuet al. proposed an ELISA based method for the detection of hepatitis B virus based on glucose oxidase (GOx)-induced fluorescence quenching of CdTe QDs (Fig. 7(a))^[94]. In this system, the GOx was coupled onto polystyrene microplates through immunoreaction of hepatitis B virus with antibody to catalyze the glucose to generate H₂O₂. The hepatitis B virus was detected by the fluorescence quenching of H₂O₂-sensitvie CdTe QDs. This method displayed high detection sensitivity, which was approximately 430-folds lower than that of HRP-catalyzed conventional ELISA. Zhouet al. reported a dual-mode rabies virus detection using dendritic silica nanospheres (DSNs) with the densely packed QDs and HRP-labeled antibody to substantially broaden the application scenarios of ELISA. DSNs were prepared for QDs encapsulation and then covalently coupled with HRP-labeled antibody. As shown inFig. 7(b), the sandwich-structure immunocomplexes were mainly formed between rabies virus antigen and antibody. After addition of H₂O₂ and TMB, an obvious color change was observed due to the HRP-catalyzed TMB oxidation^[95]. Furthermore, rabies virus can also be detected by measuring the fluorescence intensity of QDs embedded DSNs. This method displayed wider linear range and nearly 2 orders of magnitude lower than that of the conventional ELISA. This method using DSNs for the colorimetry-based prescreening and fluorescence-based quantitative detection might pave the way for virus-related disease diagnosis. Enzyme labeling is usually used for signal amplification in ELISA system, but its stability is relatively low and its activity is vulnerable to environmental interference. To solve these limitation, Zhaoet al. developed a DNA label method for immunoassay of HIV-1 p24 antigen (Fig. 7(c))^[96]. In this method, the DNA was conjugated onto indium tin oxide (ITO) electrode after the sandwich immunobinding, which can be released after treating with NaOH. The subsequent depurination of the oligonucleotide strands by H₂SO₄ can enable the oxidation of free nucleobases (poly G) at a CdTe QDs modified ITO transducer to produce the photoelectrochemical signal. The DNA labeling displays better stability, although the sensitivity needs to be further improved.

Magnetic bead-based immunoassay (MBIA) is used to modify the MB and reporter with antibodies respectively, promote their binding with the target to form MB-antibody-target-antibody-reporter complex and remove the various impurities through the magnetic separation for signal detection. Joet al. constructed QDs-embedded silica nanoparticles, which showed 500-times stronger fluorescence in comparison to individual QDs (Fig. 7(d))^[97]. In the present of target avian virus, antibody modified QDs-embedded silica nanoparticles were bound with antigen and antibody modified MBs, and the fluorescence intensity of QDs-embedded silica nanoparticles can be used for avian virus detection after the magnetic separation. The sensitivity of this MBIA method is about 2000 times better than the hemagglutination method (a typical method for influenza virus detection). In addition, Park’s group developed an innovative localized surface plasmon resonance (LSPR)-amplified magnetofluoroiimmunoassay for norovirus and influenza virus detection to further enhance the sensitivity (Fig. 7(e))^[98]. Au NPs and MBs hybrid nanocomposite (AuNP/MNP) was synthesized and used for the magnetic separation and high localized surface plasmon resonance (LSPR) effect. In the present of target norovirus, antibody modified QDs could bind with the target norovirus as well as antibody modified AuNP/MNP, and the fluorescence of QDs was effectively enhanced through the LSPR energy transfer between AuNP/MNP and QDs after the magnetic separation. However, due to magnetic separation and high LSPR effect, this method could display high signal-to-noise, which is10²-fold greater that of above ELISA. Digital immunoassays can divide analytes into microwells or emulsion droplets for independent signal amplification, thus resulting in sensitivity increase over traditional immunoassays^[99]. Wu et al proposed a digital single virus immunoassay for the multiplex virus detection using fluorescent magnetic multifunctional nanospheres (FMNs) as both the capture carriers and signal labels, which prepared by the assembling MB and QDs through electrostatic interaction (Fig. 7(f))^[100]. After sandwich immunoreaction, fluorescence images of 500 micropores were obtained. Furthermore, corresponding to with or without target virus, the micropores with or without fluorescence were denoted as “1” or “0” based on the digital analysis, respectively. The virus concentration can be calculated by the “1” probability. Green, yellow, and red fluorescent magnetic multifunctional nanospheres were used for H9N2, H1N1, and H7N9 avian influenza virus detection, respectively. This method displayed high sensitivity (0.02 pg/mL of detection limit), good specificity, and versality for multiplex detection.

LFIA is rapid easy to operate, low-cost, and point-of-care testing tool for virus infection diagnosis^[101]. In conventional LFIA, capture and control antibodies are immobilized on the test as well as the control lines in the strip, and in the present of target antigen, antigen-antibody-conjugated Au NPs complexes are formed and could pass through the test line to generate the test signals by interacting with the capture antibody. Free antibody-conjugated Au NPs could then bind with the control antibody to verify the availability of the test strip. Due to high stability, easy preparation, and strong colorimetric signal, Au NPs have been commonly used as LFIA reporter. QDs have replaced the Au NPs as the new reporter and this modification is being considered as promising strategy to enhance the detection sensitivity of LFIA. Wang’s group developed a high-performance QDs nanobead fabricated by absorption of multilayers of QDs onto the SiO₂ surface through electrostatic interaction of PEI-SiO₂/PEI and QDs to produce Si@TQD. The detection antibody was modified onto the Si@TQD, which was used for the detection of SARS-CoV-2 antigen and influenza A virus (Fig. 7(g))^[102]. This method displayed 2 orders of magnitude improvement in sensitivity compared with conventional Au NP-based LFIA. In another study, to enhance the repeatability and accuracy of LFIA, Hanet al. fabricated a dual-functional SiO₂@Au/QD fluorescence labels by coating Au NPs and QDs onto SiO₂. The detection antibody was modified onto SiO₂@Au/QD, which was used as both colorimetric and fluorescence signals for SARS-CoV-2 spike protein detection (Fig. 7(h))^[103]. The colorimetric signal was employed for the visual detection and rapid screening of SARS-CoV-2 infection, whereas the fluorescence signal was used for sensitive and quantitative detection of virus infection at the early stage, which displayed good repeatability, accuracy, and broad application range. Portable instruments to read the QDs fluorescence signal are highly in demand for providing diagnosis at home, while there are still some challenges to be solved, including portable excitation light source, and simple and accurate fluorescent reading system.

Because no separation and purification procedures are required, homogeneous immunoassays can enable a one-step, rapid, and direct detection of different analytes. Homogenous immunoassays are mainly based on the fluorescence resonance energy transfer (FRET) from antigen/antibody modified QDs to quencher, which could be blocked due to antibody-antigen interactions-induced distance enlargement between the donor (QDs) and acceptor (quencher). A variety of molecules have been used as acceptors, such as metal-ligand complex, GO, and Au NPs. Chenet al. developed a homogeneous immunoassay for enterovirus 71 using Ru(bpy)₂(mcbpy-O-Su-ester)(PF6)₂-antibody (Ru-Ab) as the quencher of QDs fluorescence and the capture probe of the target enterovirus 71 (EV71). As shown inFig. 8(a), Ru-Ab can bind with QDs through the electrostatic interaction and quench the QDs fluorescence^[104]. The QDs were then set free and the fluorescence was recovered after the binding of Ru-Ab with target EV71. This method could not only provide rapid and precise quantitative determination of EV71 by fluorescence intensity but also could be applied in semi- \quantitative EV71 detection by digital visualization, which could yield a simple, fast, highly selective approach for virus infection diagnosis. In another report, combining the wide absorption range of GO and multiplex emission of QDs, Chenet al. proposed a method for EV71 and coxsackievirus B3 (CVB3) simultaneous detection using antibody modified QDs and GO (Fig. 8(b))^[105]. Green and red QDs were modified with EV71 antibody and CVB3 antibody, both of which were quenched by GO through π–π stacking interaction of antibody and GO. After adding the target virus, the interaction of antibody modified QDs with GO was broken up, and the fluorescence of QDs was recovered. Due to the broad excitation and narrow emission of QDs, this sensor was used for the multiplex detection using a single-wavelength excitation light and can provide a novel option for screening of virus species or subtype differentiation both simply and effectively. Nasrinet al. developed a method for norovirus detection by combining LSPR behavior of Au NPs and FRET from QDs to Au NPs to markedly improve the detection sensitivity (Fig. 8(c))^[98]. Without target virus, QDs fluorescence was quenched by Au NPs via FRET due to the close interaction of QDs and Au NPs. However, in the present of target virus, steric hindrance blocked the FRET from QDs to Au NPs, and further triggered the fluorescence enhancement of QDs via target-induced the LSPR from Au NPs to QDs. This method provided synergistic fluorescence enhancement of QDs by combining FRET and LSPR, which displayed pretty high sensitivity which was 100-fold greater than that of commercial ELISA kit.

Zhanget al. reported a novel method for the detection of parvovirus antibody by QDs-loaded spore-based monodisperse microparticles (QDSMs) to improve both output and sensitivity (Fig. 8(d))^[106]. Antigen modified green QDSMs were constructed by embedding QDs onto the surface of spore-based monodisperse microparticles and modifying with parvovirus. In the presence of the target antibody, antigen modified green QDSMs were bound with the target antibody and Alexa Fluor 647-IgG to form QDSM-detection antibody-Alexa Fluor 647-IgG, which were used as model system to detect the parvovirus antibody via using flow cytometry. A variety of barcodes can be obtained by potential interaction of QDSMs with the distinct colors upon using emitting fluorescence dye, which displayed enormous potential in high-throughput immunoassays. Furthermore, due to the advantages such as simplicity, low cost, high throughout and eco-friendliness, this method may find wide applications in rapid disease screening. Recently, many homogeneous immunoassays based on the nucleic acid amplification and protein complementation assays have been developed, such as proximity ligation and split tripart Nanoluciferase^[107]. Liet al. reported an ultrasensitive CRISPR-based antibody detection (UCAD) assay that can effectively translate the detection of anti-SARS-CoV-2 antibodies into CRISPR-based nucleic acid detection by combining proximity ligation, T4 polymerase-based amplification, and RPA-assisted CRISPR/Cas12a system. As shown inFig. 8(e), DNA modified detection antibody and DNA modified region binding domain were found to bind with anti-SARS-CoV-2 antibodies to induce the proximity binding of DNA probes and the formation of dsDNA assisted by T4 polymerase^[108]. Recombinase polymerase amplification of dsDNA can cause amplicon to trigger the subsequent indiscriminate single-stranded DNase activity of Cas12a to produce the fluorescence signal. This method could translate antibody into the nucleic acid amplification, which displayed 10,000 times more sensitive than the commercial ELISA and could be used for rapidly tracking the antibody levels changes with ultrahigh sensitivity in infected or vaccinated individuals. Integrated with QDs, these methods could provide higher sensitivity and better portability due to the high quantum yield of QDs.

This review article mainly discussed the strategies employed for the synthesis and biofunctionalization of QDs for virus detection. The Covid-19 pandemic caused by the virus has attracted widespread attention around the world. The traditional virus detection method is performed in the laboratory by using a large clinical analyzer, such as PCR and ELISA. These methods require the availability of trained technicians and long detection time. The rapid development of QDs has significantly improved the design and manufacture of novel devices for biosensing. A variety of methods based on QDs for virus detection to substantially simplify operation and enhance detection sensitivity, including nucleic acid hybridization, nucleic acid amplification, probe signal enhancement, and enzyme linked amplification have been developed. However, several challenges must be addressed to facilitate their better performance in controlling the outbreak of pandemic diseases. 1) The potential toxicity of the semiconductor QDs can effectively limit their clinic applications in diseases diagnosis. Hence, the preparation of relatively less and even non-toxic QDs are required for the construction of safe and eco-friendly biosensors. In addition, some lab-made immature QDs are hardly produced at large-scale, so it is necessary to optimize simple and middle method for preparation of large-scale QDs. 2) For the viral nucleic acid detection, conventional PCR or LAMP are time-consuming, whereas application of the new emerging technology, such as CRISPR, is associated with high cost. In addition, viral nucleic acid extraction is unavoidable and complex. The combination of the nucleic acid free-extraction method and QD-based virus biosensor could significantly improve the operability of molecular diagnosis. 3) For immunoassays, ELISA is time-consuming, LFIA has low sensitivity, however there only are few effective methods available that can integrate both high speed and sensitivity. Some synthetic biology sensors have been developed that can exhibit rapidity, specificity, as well as robustness and thus integrating them with QDs as reporter could further enhance the detection sensitivity.