Human-infecting pathogens that transmit through the air pose a significant threat to public health. As a prominent instance, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that caused the COVID-19 pandemic has affected the world in an unprecedented manner over the past few years. Despite the dissipating pandemic gloom, the lessons we have learned in dealing with pathogen-laden aerosols should be thoroughly reviewed because the airborne transmission risk may have been grossly underestimated. From a bioanalytical chemistry perspective, on-site airborne pathogen detection can be an effective non-pharmaceutic intervention (NPI) strategy, with on-site airborne pathogen detection and early-stage infection risk evaluation reducing the spread of disease and enabling life-saving decisions to be made. In light of this, we summarize the recent advances in highly efficient pathogen-laden aerosol sampling approaches, bioanalytical sensing technologies, and the prospects for airborne pathogen exposure measurement and evidence-based transmission interventions. We also discuss open challenges facing general bioaerosols detection, such as handling complex aerosol samples, improving sensitivity for airborne pathogen quantification, and establishing a risk assessment system with high spatiotemporal resolution for mitigating airborne transmission risks. This review provides a multidisciplinary outlook for future opportunities to improve the on-site airborne pathogen detection techniques, thereby enhancing the preparedness for more on-site bioaerosols measurement scenarios, such as monitoring high-risk pathogens on airplanes, weaponized pathogen aerosols, influenza variants at the workplace, and pollutant correlated with sick building syndromes. The framework for on-site airborne pathogen detection and transmission risk mitigation. Therefore, we focus our discussion on the development of rapid and bio-integrable pathogen sampling devices, especially those that meet the above requirements.19For on-site pathogen detection, the effective collection of bioaerosols is a critical prerequisite, 41–44 In a manner different to summaries of existing personal diagnosis approaches, we primarily focus on reviewing novel biosensing systems that can rapidly detect trace amounts of airborne pathogen particles on-site. Ideally, on-site biosensing devices should be: (1) Sensitive, with superior limit of detection to quantify trace amount of airborne pathogens; (2) accurate and specific, to provide reliable pathogen exposure information and avoid background interferences from the complex aerosol matrix; (3) robust, so that they can operate on-site in different environments; (4) swift, to provide fast quantitative biochemical analysis results; (5) versatile, allowing the detection of multiple or different pathogen targets under various scenarios; (6) automated, enabling continuous monitoring with minimal human operations; (7) connectable, allowing access to Internet of Things (IoT) for data analysis by edge computation, remote monitoring, telecommunication, and networking. We review and summarize the bioanalytical chemistry technologies that can meet or have the potential to meet the above “SARSVAC” criteria. The potential interferences of typical airborne pollutants for on-site pathogen detection as well as the feasible solutions are also discussed. Furthermore, early warning via biosensing systems necessitates detection in public areas, where trace amounts of airborne pathogens potentially exist. The last section of this review is dedicated to inspecting novel “plug-and-play” biochemical amplification strategies that help to improve the sensitivity, accuracy, functionality, and robustness of on-site airborne pathogen detection. Last but not least, we share our insights on the open challenges in on-site airborne pathogen detection, such as handling complex aerosol samples, improving the sensitivity for detecting trace amounts of airborne pathogen, and establishing high-spatiotemporal risk assessment systems for mitigating airborne pathogen transmission risk. We hope our cross-disciplinary outlooks in this review could provide inspiration for researchers from different research communities so as to further improve the preparedness for on-site airborne pathogen quantification and risk assessment.Similar to bioaerosol sampling devices, many advanced on-site and point-of-care biosensing approaches have been proposed recently.22.145,46 One of them was a “droplet”-based infection, which refers to large respiratory droplets that are rapidly removed from the air by gravity before drying. These droplets generally settle on the surface of objects and indirectly infect susceptible individual (infectee). The second form was introduced as “aerosol”-transmitted infection. Generally, the term “aerosols” represents the dried residues or droplet nuclei suspended in the air. Since the sizes of aerosols are typically small (<5 μm), their transmission depends dominantly on the buoyancy of the air, allowing them to remain transporting for extended periods of time and longer distances compared to the droplets. Airborne transmissible pathogens could be directly released from an infected person (infector) and transmitted to an infectee via these two airborne-based infection routes . Traditionally, it is widely believed that direct contact, large respiratory droplets, and droplet-contaminated surface (fomite) are the dominant transmission approaches for respiratory viruses, while long-distance airborne transmission (aerosol) only occurred in special circumstances and for specific pathogens. via aerosols. For example, the fecal coliform group bacteria such as Escherichia coli, Salmonella enterica species, Enterococci species, Cryptosporidium parvum, and Campylobacter species can transmit in air and infect others through the aerosolization of contaminated wastewater. Meanwhile, fungal bioaerosols, which consist of spores, mycelium fragments, and debris, can be inhaled and cause numerous symptoms including allergies, irritation, and opportunistic infections.47 There are also a number of studies demonstrating the aerosol transmission of respiratory viruses, including influenza, rhinovirus, adenovirus, measles virus, respiratory syncytial virus (RSV), and human coronavirus .5–7,48,49 For instance, a large community outbreak of SARS-CoV in Hong Kong was investigated by applying airflow dynamic modelling, in which the infection data and the predicted 3D spread results were well correlated.7 This study highlighted the importance of considering the aerosol transmission route in infection prevention and control. In addition to respiratory infections and epidemic diseases, bioaerosols can impact the quality of the air we breathe, thereby affecting our daily well-being. The inhalation of bioaerosols could cause irritation and inflammation in the respiratory system. Consequently, sick building syndrome (SBS), characterized by coughing, wheezing, shortness of breath, headaches, fatigue, and decreased cognitive function, may occur in enclosed indoor environments.50Dating back to the 1930s, airborne-based infections and transmissions have been classified into two distinct forms, mainly depending on the size of the airborne pathogen carriers.11,34,51,52 Although long-distance transmission and potential high infectivity have been recognized, there are still some controversies about the airborne pathogen transmission, largely because of an incomprehensive understanding of the pathogen-laden aerosols and insufficient reliable analytical chemistry techniques in the toolbox for on-site bioaerosol detection.28,53–55 Currently, the accumulated research results not only illuminate the limitation of conventional point-of-views about airborne virus transmission but also bridge the cognitive gap and serve as important knowledge supports for the emerging research regarding on-site bioaerosol sampling, transduction, and risk level assessment. By systematically sorting out the characteristics of aerosol transmission in the following sections, we are aiming to identify the internal physiochemical properties of pathogenetic bioaerosols and the external potential environmental factors that may impact airborne transmission. More importantly, these characteristics can facilitate the development of on-site bioaerosol testing systems for reliable risk assessment and further deepen the epidemiological knowledge about pathogenetic bioaerosol transmission.In the past few years, the characteristics of bioaerosol transmission have been extensively studied, and many characteristics of the airborne pathogen transmission have been clearly elucidated. Pathogens such as SARS-CoV-2 can be transmitted in aerosols in different environmental scenarios such as health-care settings, private cars, public transportation, schools, bars, and gymnasiums.2.256 Airborne bacteria can vary in size depending on the species and typically falls between 0.3 and 60 μm in diameter  generated in the lower respiratory tract were likely to contain more respiratory viruses than larger aerosols (>5 μm) generated in the upper respiratory tract.62,63For transmittable pathogenic bioaerosols that cause infections in the respiratory tract, expiratory activities such as breathing, speaking, coughing, sneezing, and singing could be one of the initial sources .10,57,5864 One recent work suggested that post-symptomatic individuals with coronavirus infection produced higher number of exhaled aerosols at about 1300–2700 aerosol particles per liter in air (ap per L), while the noninfected control and recovered individuals expelled aerosols with much less number concentrations at 7–198 ap per L.64 Other expiratory activities such as speaking, coughing, and sneezing can produce even higher number of airborne particles. For instance, loud speech can emit more than 1000 oral airborne particles per s, which refers to a number concentration greater than 2600 ap per L .10The number concentration of airborne pathogen is another key physiochemical characteristic that should be considered for disease transmission control. However, the number concentration of exhaled pathogen-laden aerosols have large interindividual variability by almost three orders of magnitude and heavily depend on the disease stage, age, and preexisting health conditions.65 Evaporation can decrease the diameter of airborne particles, as shown in 66 For instance, a 100 μm bioaerosol at a height of 1.5 m can remain in the air for just 5 s, while a smaller bioaerosol with 1 μm diameter can be suspended for more than 12 h.66 One recent time-resolved modelling study demonstrated that, as shown in T = 20 °C and RH = 60%).67 Other intrinsic physical properties, such as the initial velocity and the aerosol morphology, also directly determine the transport distance and behaviors in air, as shown in −1, thereby spreading the virus over a significant distance.68,69In case of the bioaerosol dimension and number concentration, it is important to consider that the size distribution of pathogen-laden aerosols could evolve over time due to the in-air evaporation, fusion, condensation, and deposition.70 The initial airborne droplets or aerosols could have similar chemical compositions as the respiratory fluid, which includes non-volatile ions, proteins, surfactants, and organic substances, as shown in 71 Therefore, the physicochemical impacts on the viability of airborne pathogens should be considered when using the on-site measurement results to assess the transmission risk. Section 2.4 will discuss the external environmental factors that could potentially impact the viability of airborne pathogen and the quantitative on-site detection results.The alterations of physiochemical properties during the generation and transportation of bioaerosols can also impact the viability and infectivity of airborne pathogens.2.3via air through inhalation. Typical scenarios and case studies of airborne pathogen transmission, including virus, bacteria, and fungi, are summarized in Table S2  systems. Among these, the inhalation of respiratory bioaerosols containing the pathogen is the most prevalent and dominant transmission route. Previous studies have demonstrated that airborne pathogens such as tuberculosis, measles, chickenpox, and influenza can be transmitted le S2 ESI.72 Reportedly, the HVAC system also contributed to the outbreak of Legionnaires' disease in different indoor environments.73,74 In these cases, the air-conditioner was suspected to be the main driving factor to promote the aerosol transmission and potentially caused the sick-building syndrome.Ventilation is an effective means of addressing and reducing the indoor airborne transmission of infectious pathogens. Increased airflow can effectively reduce the concentration of bioaerosols in enclosed spaces. However, external airflow such as HVAC systems may facilitate the diffuse and disperse of airborne particles in the environment, particularly fine pathogen-containing aerosols. For example, a cluster of SARS-CoV-2 infections was reported among three families seated at adjacent restaurant tables.75 Moreover, animal farms have been considered as the critical reservoir of antibiotic resistant bacteria (ARB) and potentially spreading antibiotic resistant gene (ARG) that threaten human and animal health worldwide.76Additionally, alternative bioaerosol transmission routes based on environmental sources may also widely exist. Pathogens may also be present in the soil, water, or animal waste. These pathogens may become airborne through natural processes such as wind or human activities, such as construction or farming.E. coli, Clostridium difficile, influenza, SARS-CoV-2, Mycobacterium tuberculosis, and Streptococcus pneumoniae. Recent studies have revealed that the amount of SARS-CoV-2 viral genetic materials were found to be 102–105 copies per mL in urine and 102–107 copies per mL in stool samples.77–79 One investigation also suggested that a large number of aerosols in the size range 0.3–3 μm were generated during toilet biomatter flushing, and these fine aerosols could remain suspended in air for long periods of time and reach heights of >1.52 m.77 A fluidic dynamic simulation also demonstrated that up to 60% of pathogen-laden aerosols can transport be upward above the toilet seat during flushing.79 These discoveries also indicated the potential airborne transmission route from sewage networks and wastewater treatment.80The human digestive system can also excrete infectious pathogens such as 10 Similarly, another recent study also suggested that 10 μm aerosols would take 9 min before reaching the ground when produced at a height of 160 cm.81Another important aspect that needs to be considered in the airborne transmission route is the lifetime of airborne pathogens, which is dominantly determined by two factors: (1) the airborne lifetime that the bioaerosols remain suspended in air and (2) the inactivation lifetime  determined by external environment factors. The airborne lifetime is related to the stabilized aerodynamic size. As mentioned earlier, the suspension duration can be predicted theoretically by Stokes' law, where the terminal velocity of a falling aerosols is approximately proportional to the square of its diameter. In a recent study, the authors utilized a stagnant-air environment to investigate the air-suspension lifetime of aerosols produced by speech. Apart from the time-resolved simulation showed in 82 It has been revealed that the median half-life of SARS-CoV-2 was estimated to be about 1.1 h at 65% relative humidity (RH) and 21–23 °C.83 In another experimental study, airborne SARS-CoV-2 virus particles retained infectivity and virion integrity for up to 16 h at 53% RH and 23 °C.84 The half-life and infectivity of airborne pathogens are predominantly affected by ambient environmental conditions such as RH, temperature, and irradiation conditions.85 These key environmental and external factors affecting the airborne transmission of pathogens are discussed in detail in the following section.Regarding the inactivation lifetime of airborne pathogens, the Goldberg-drum approach is typically used to investigate the viability and persistence of the suspended pathogens.2.42.4.186 Therefore, airborne transmissibility can be affected by ambient temperature in many different aspects such as temperature-dependent infectivity and airborne viability. Bacteria have a range of optimal temperatures for growth, and any deviation from that range can negatively affect their viability. One recent study investigated self-decay laws and efficiencies of airborne bacteria under different temperatures.87 As illustrated in 88 At a typical room temperature of 24 °C, the half-life of the virus ranged from 6.3 to 18.6 h but significantly decreased to 1.0 to 8.9 h when the temperature increased to 35 °C. The thermodynamic nature of biomolecules may decay faster at elevated temperatures, thereby making pathogens lose their viability and infectibility. Therefore, airborne pathogens such as coronavirus and influenza virus are more persistent at lower temperatures.Temperature is significant in maintaining the viability of the airborne pathogens in terms of biomolecular morphology, structure, and function.89 Specifically, the average ambient temperature of the lower respiratory tract is approximately 35–37 °C under normal quiet breathing conditions and slightly decreases to about 32–33 °C in the upper trachea and mouth. By an increase in the breathing rate, the temperature along the airway may slightly fell to 33.9 °C and 29.2 °C, respectively. A recent study has shown that the infectious titer and replication kinetics were higher at lower temperature of 33 °C than that at 37 °C, which indicated that respiratory viruses like SARS-CoV-2 had a better replication capacity in the upper respiratory tract.90 Therefore, colder incubation temperature, such as the elevated breathing rate and lower environmental temperature, allowed the virus to replicate faster, therefore facilitating the transmission of the airborne viruses.The second aspect affecting airborne pathogen transmission is the infectivity under different ambient temperatures. During expiration, the temperature along the respiratory tract progressively decreases with airflow from the lower to the upper part.2.4.291 In contrast, at a high RHs of 80% and above, the airborne virus might be far more stable, with infectivity rarely falling below 80% after 2 min. Regarding the long-term infectivity, a decrease to ∼10% of the starting value was observable for both RH = 40% and 90% over 20 min. In addition, different chemical components in the original aerosols or droplets may also affect the stability of airborne pathogens when changing the RH. An acidic environment may decrease the pathogenic viability, while organic macromolecule contents  could enhance the longevity of airborne viruses.91,92 Based on the experiments results from the literature, Aganovic and co-workers summarized the biological decay constant (k) at different RHs from 20% to 90% at a constant temperature of 20 °C, as shown in 93 The airborne enveloped viruses demonstrated a high viability and a low decay rate at RH = 37%. To date, the mechanism and impact of RH-induced inactivation are still not fully understood. The efflorescence-deliquescence divergent infectivity (EDDI) hypothesis was recently used to predict the RH-dependent survival of airborne virus, which suggested that the surviving fraction in low RH was higher than that in higher RH aerosols.94 This trend was also consistent with the collective experimental results shown in Typically, pathogen-laden aerosols expelled from respiratory tract lose both heat and moisture, thereby quickly forming smaller equilibrium particles within seconds . The evaNotably, enveloped airborne viruses such as influenza and coronavirus follow this trend and survive longer at lower RHs, whereas non-enveloped airborne viruses have a tendency to survive longer at higher RHs. Similarly, both Gram-positive and Gram-negative bacteria have an optimal RH range between 40% and 60% for their survival. High RH levels can cause the cell wall to rupture, leading to bacterial death. On the other hand, low RH levels can cause the cell wall to dehydrate, leading to a decrease in bacterial growth and reproduction. Therefore, it is important to consider both the pathogen species and its behaviors under different RHs when assessing the transmission risk of airborne pathogens.2.4.385,95 In airborne transmission, bioaerosols are inevitably exposed to different irradiations such as natural sunlight, especially in outdoor environments. Using the solar simulator with spectra designed to represent the UV range (280–400 nm) of natural sunlight, the decay rate and lifetime of airborne bacteria and viruses were investigated in a Goldberg rotating drum aerosol chamber.95 For enveloped viruses such as influenza and SARS-CoV-2, the mean decay rate in simulated saliva were found to be 0.121 ± 0.017 min−1  under winter sunlight condition and 0.306 ± 0.097 min−1  under summer sunlight condition. The mean decay rate without simulated sunlight was much lower at 0.008 ± 0.011 min−1 . In addition, prolonged exposure to visible light of short wavelengths (400–420 nm) may also reduce the viability of airborne pathogens.96 Additionally, when applying an average UV intensity of 10 W cm−2, 63 percent of airborne tuberculosis bacteria could be inactivated in 24 s, and 99 percent were eliminated in 2 min. Another study demonstrated that the median natural inactivation ratio of airborne Mycobacterium abscessus was 62.2% after 30 min and 75.5% after 40 min.97 An increased UV-c radiation dose by 83.1 μW s cm−2 can elevate the ratio to 83.1%.Radiations including the UV light can rapidly inactivate pathogens in aerosols.Therefore, the further evaluation of the surrounding UV (radiation) exposure levels is recommended in order to accurately calculate the viability and actual transmission risk of pathogens. Although ordinary indoor lighting systems, such as white LED and Halogen lamp, barely contain radiation in the UV band, the decay rate of the airborne pathogens during on-site risk assessment should also be further investigated and considered.2.4.498,99 The general inactivation mechanism is based on chemical reactions between the oxidants and the biomolecules constituting the essential structures or functions of pathogens.100 For instance, ozone or other oxidant species, e.g., hydroxyl radical, singlet oxygen can react with the nucleic acids of the airborne pathogens and potentially impact the accuracy of biomolecular sensing results. The recent experimental results demonstrated that the longevity of the enveloped coronavirus was negatively impacted by ozone.98 The spread of airborne viruses was obviously reduced by three order of magnitudes by increasing the ambient ozone concentration level from 48.83 to 94.67 μg m−3. Similar to effective UV treatment, high oxidant concentrations (129 000 μg m−3 min) may inactivate most of the airborne viruses within minutes.99,101The inactivation of airborne pathogens by ozone and other oxidizing agents is another significant factor that needs to be considered in evaluating the infection and transmission risk.102,103 Therefore, in the on-site bioaerosol sampling and sensing process, it is necessary to fully consider the inactivation and decay rate caused by different environmental factors so as to accurately calculate the concentration and quantitative infection risk level of viable pathogens.The key environmental factors mentioned above can individually impact the transmissibility and infectivity of airborne pathogens. However, the overall impact on the airborne transmission should be carefully considered based on the specific on-site environments. For instance, although both higher RHs and UV radiation can inactivate airborne enveloped virus, the elevated RH may also reduce the UV damage and thereby increase their persistence.2.5Over the past few decades, airborne pathogens and bioaerosols have been a growing public health concern due to their potential to cause widespread disease outbreaks, their use as biological weapons, and daily health impact such as sick-building syndrome.One of the main concerns is the airborne transmission of contagious pathogens. Based on the physiochemical properties of bioaerosols introduced in Section 2.3, airborne pathogens transport and deposition in the respiratory airways can be determined using several established models such as the multiple-path particle dosimetry (MPPD) model, as shown in Streptococcus pneumoniae, a 0.5–1.2 μm bacterium that can penetrate into the trachea, bronchi, and cause pneumonia. Another example is Mycobacterium tuberculosis, the bacterium that causes tuberculosis. Viruses such as influenza and SARS-CoV-2 can also cause lower respiratory tract infections. Most seasonal Influenza viruses infect the cells lining in the larger airways, trachea, and nasopharynx, while the pneumotropic influenza A virus strains such as H5N1 target the lower respiratory tract like the lung airways and alveoli.104 Additionally, SARS-CoV, MERS-CoV, and SARS-CoV-2 also replicate in the lower respiratory tract in humans. By considering the dimension of airborne pathogens, fine (<2.5 μm) and ultrafine (<0.1 μm) bioaerosols have become a common and growing concern due to their large penetration depth and lower inoculum in causing respiratory tract infections. However, current bioanalytical systems for detecting bioaerosols are still not well-established. This has also led to a current technology shortage in the toolbox for epidemic prevention and public health intervention.Regarding normal respiratory activities, large droplets mainly transport for a short distance in the air , but they deposit on the lip/eye/nostril mucosa of another person at close contact and cause inoculation. In contrast, pathogen bioaerosols smaller than 2.5 μm can remain suspended in the air for a long time and penetrate deeply into the lower respiratory tract to cause infections. One example is Additionally, bioaerosols and airborne pathogens may influence our daily lives in a pervasive but covert manner. The accumulation of airborne microorganisms may result in sick building syndrome (SBS), characterized by asthma symptoms, mucous membrane irritation, gastrointestinal disturbances, and neurotoxic effect. Severe toxicosis and cancer could also result from a long-term and continuous exposure to airborne mycotoxin. At present, the specific conditions that lead to SBS are not fully understood. All of these concerns about airborne pathogens motivate ongoing research into the detection and risk evaluation of bioaerosols.336,105,106 To achieve reliable risk level determination of airborne pathogen transmission, many key factors and aspects need to be considered, such as the airborne pathogen emission, airborne pathway by considering the decay and removal rate, human exposure, and dose–response relationships.105,107,108 Using respiratory viruses as examples, this section will review the recent advances and challenges of risk assessment of airborne pathogens based on the extensive and systematic studies conducted in recent years.Based on the progressively improved understanding of airborne pathogen transmission, the importance of the fast and quantitative risk assessment of airborne pathogen transmission for a wide variety of public and indoor environments has been recognized.3.1.Airborne pathogen exposure is defined as a contact over time and space between a person and bioaerosols. Exposure assessment is to identify and define the exposures that occur or are anticipated to occur within a specific microenvironment. This can be a complex endeavor requiring analysis of many different aspects of the contact such as bioaerosol emission, removal rate, exposure pathway and route, and exposure concentration and duration.e.g., on-site and point-of-contact sensing) and indirect measurement . Direct measurements are an unequivocal way to establish individual exposure assessment to a specific airborne pathogen like coronavirus, influenza, or Bacillus anthracis. However, the direct measurement of airborne virus exposure in this ongoing COVID-19 remains technically impracticable and unaffordable for broad applications. Thus, using mathematical models and information abstracted from physical realities can be a cost-effective way for the urgent need of extensive exposure assessment and risk level determination. Some representative measurement results  and preliminary characteristics can be used in the models for exposure estimations. Therefore, the main challenge is to develop appropriate and robust mathematical models so as to extrapolate the exposures and doses of individuals and communities in more different environmental scenarios. In this section, recent advances in bioaerosol exposure and transmission risk assessment will be analyzed using SARS-CoV-2 as a typical case study.As the central feature of airborne transmission risk assessment, quantitative exposure assessment can be approached in two general ways: direct measurement  and influenza viruses were directly measured in the exhaled breath and coughs of patients with acute respiratory illness.105 The virus (quanta) emission rate is evaluated asCv is the viral load in the sputum , Ci (quanta RNA copies−1) is a conversion factor defined as the ratio between one infectious quantum and the infectious dose expressed in viral RNA copies, IR is the inhalation rate (m3 h−1), and Vd is the droplet volume concentration expelled by the infectious person (mL m−3). In this work, a quantum is defined as a dose of airborne droplet nuclei required to cause infection in 63% of susceptible persons. Based on the retrospective results on human coronavirus, the authors adopted an average CPFU value (quanta-to-plaque forming unit conversion factor) of 2.1 × 102 PFU quanta−1 and an average CRNA (RNA copies-to-PFU conversion factor) of 1.3 × 102 RNA copies PFU−1 to quantify the emission rate.110,111Recently, Buonanno and co-workers proposed a forward emission approach to estimate the airborne virus emission based on the viral load in the sputum.112,113 Additionally, the majority of respiratory pathogens lack systematic research efforts comparable to those of the novel SARS-CoV-2 virus. Consequently, there are no reliable data to evaluate the exhalation discharge of pathogens at the individual level.Emission estimation is a convenient but indirect method for predicting the airborne pathogen concentrations. Although some different factors have been considered, the amount of pathogen shedding in patients can fluctuate greatly depending on the personal conditions such as the vaccination status, stage of infection, personal health conditions, and the infected coronavirus strain.3.1.2i.e., jet models, box models, and computational fluid dynamics (CFD) models, as shown in 107,114 Jet models are utilized for exposure at a short distance, while the box models are usually for exposure assessment in the enclosure space with the assumption of a well-mixed condition with the homogenous distribution of particles. CFD models represent the detailed physical models for both short- and long-range dispersion.107There are mainly three types of numerical models to evaluate the exposure to bioaerosols, 115,116 Turbulent round jet models are usually adopted to describe the velocity profiles in the expired jets, which normally follow Gaussian profiles.117 Lagrangian methods, e.g., discrete random walk model, are adopted to estimate the motion of the particles in the jets.115 The simplified jets and dispersion model could be utilized to evaluate the exposure over short distances, e.g., several meters, due to the combined impacts of large droplets and small airborne droplet nuclei.For the exposure due to close contact, mathematical models for the dynamics of expired jets are normally utilized to evaluate the exposure to large droplets (>5 μm) and small airborne droplet nuclei (<5 μm) in the exhaled plume .115,116 e.g., rooms, cars, and airplanes.107 Box models assume well-mixed contaminants and homogeneous concentrations in the enclosure space; thus, the variation of the airborne concentration of active pathogens can be described by an ordinary differential equation based on mass balance, which is solved analytically.35,107,118,119 Box modes are dependent on the important parameters, including pathogen shedding rate (emissions), the inner volume of enclosure space, ventilation rate, deposition of particles on the surfaces, and biological decay.119 Parameterization models are normally required for the estimation of deposition rates.120 Box models could quantify the average exposure in the enclosure space, which is a reasonable assumption when people tend to move around, e.g., in supermarkets and shopping malls.121 However, it might have biases when people are relatively static, e.g., in classrooms and libraries, where the spatial distribution of bioaerosols like coronavirus becomes more important, and detailed methods are needed.122Box models, as shown in 114,123–129 CFD models are also used to investigate the transport of pathogen bioaerosol in the respiratory airways, which could contribute to better exposure assessment.130 In the CFD models, the Eulerian–Lagrangian-coupled framework has been normally adopted respectively for flow field (Eulerian method) and particle motion (Lagrangian method). The effects of drag force, gravity, and lift force on particle motion are considered in the Lagrangian method to simulate discrete aerosol particles.Computational fluid dynamics (CFD) models, as shown in 3.250, which refer to the estimated number of pathogens required to produce infection in 50% of normal adult humans, is given in 131The dose–response relationship depicts the possibility of infection or other outcomes of disease given the exposure to a certain number of pathogens including virus and bacteria. It is the indispensable component in risk assessment to link the exposure level and infection risk. The ID3.2.1e.g., mice, ferrets, and monkeys) are usually conducted to investigate the infectious dose. Animal models are developed by transgenic methods to express the human receptor for pathogens and to simulate the infection of humans.Ethical concerns limit the investigation of infection by challenging healthy volunteers, especially for pathogens with high mortality. As a result, animal studies (3 PFU for ferrets and 1.8 × 103 PFU for hamsters), which was considered close to the common infection conditions in humans.132 Three ferrets had lethargy on days 7 and 8 post-infection. On days 2 and 4 post-infection, viral RNA was found in the trachea from 2 out of 3 ferrets, but no viral RNA was detected in the lung. Viral RNA was observed in the trachea and lung from 3 out of 3 hamsters. The low challenge dose induced consistent lung infection for hamsters but not for ferrets. In another study, six ferrets were intranasally inoculated with a high viral dose of 105.5 TCID50 .133 Increased body temperature was observed in all of the ferrets on day 4 after the inoculation, and viral RNA was found in the lungs of all the six ferrets. A high intranasal infection dose might be required to induce lung infection for ferrets. Intratracheal inoculation with 106 TCID50 of SARS-CoV-2 (strain BavPat1/2020) caused 100% lung infection of nine ferrets. Similar results were obtained in a study intranasally challenging ferrets with different doses , where viral RNA shedding in the upper respiratory tract was observed in 6/6 ferrets for medium and high doses, but only 1/6 ferrets showed similar signs with low dose (500 PFU).134Recently, twelve 10-month-old ferrets and fifty hamsters were inoculated by the intranasal route with a low viral dose of SARS-CoV-2  conducted a systematic review and meta-analysis to evaluate the transmission risk of SARS-CoV-2, and the results indicated that the anticipated probability of viral infection is about 12.8% within 1 m and about 2.6% at a further distance based on the studies of the beta-coronaviruses, which include SARS-CoV and MERS-CoV.136 Using the results from meta-analysis, a simple framework was developed to integrate the a priori dose–response relation for SARS-CoV based on mice experiments and respiratory virus shedding in exhaled breath to estimate the dose–response relation for humans.137In addition to animal studies, the epidemiological investigations, systematic reviews, and meta-analysis shed light on the dose–response relation for humans.3.2.3138 The infection risk of the emitted virus-laden particles was inversely estimated based on the data from the Skagit Valley Chorale superspreading event and the Well-Riley risk equation.33The dose–response relation could also be estimated based on the case studies. Case studies were utilized to derive the dose–response probability of infection curves for foot-and-mouth disease virus (FMDV) using the infected cases and the estimated exposure rates for the farms from 1967 to 1968 at Oswestry, Shropshire, UK.3.33.3.1e.g., the distribution of exposures among members of a population) and group exposures (a population mean). The individual infection risk due to the exposure to airborne pathogens can be evaluated by the combination of exposure assessment and dose–response analysis, which is referred to as risk characterization. We tabulated the quantitative infection risks of airborne SARS-CoV-2 virus for various indoor environments in light of the research works that emerged during the pandemic  to cause infection in 63% of susceptible people based on the data for the infection of transgenic mice susceptible to SARS-CoV.110 Median viral shedding (101.5 PFU per h) was equivalent to about 8 × 10−2 quanta per h. A box model was utilized to evaluate the exposure. The results indicated that the inflection risk after 1 h of exposure with one infected person in the market was about 2.23 × 10−5 . The difference between the upper and lower bounds of the 95% confidence interval of the estimated risk was about 2 orders of magnitude, indicating significant uncertainties in the assessment due to the limited and uncertain information on SARS-CoV-2. Monte Carlo uncertainty analysis suggested that dose–response model, viral shedding, and biological decay contributed about 56.8%, 34.5%, and 8.7%, respectively, to the final uncertainty.Probabilistic risk assessment is usually adopted to quantify the uncertainties in the risk assessment and to investigate the sensitivities of the results to the input parameters. One case study for the Wuhan South China Seafood Market integrated the key processes, i.e., hospital room, gym, public indoor environments (e.g. restaurant and bank), and conference room or auditorium, were investigated.105 The median viral shedding rates were assumed to range from the lowest level of 3.7 × 10−1 quanta per h from a patient at rest in a hospital room to the highest level of 32 quanta per h from a speaking or singing person in a conference room or auditorium. One quantum was assumed to be 2.1 × 102 PFU. The exposure was estimated by a box model. The median infection risk induced by aerosol transmission with 1 h-exposure was estimated to be from 10−3 to 10−2. In another study, the magnitude of the estimated median infection risks due to aerosol transmission of SARS-CoV-2 ranged from 10−5 to 10−4 depending on room sizes and ventilation rates.137 A detailed theoretical model estimated the risk for indoor airborne transmission as about 10−3 to 10−2 with 1 h exposure and without a mask.142The infection risks in four different typical scenarios, −2 to 10−1 for 1 h exposure in typical indoor scenarios with superspreading events, including a call center in Korea and buses in Ningbo and Hunan, China.125 Wells–Riley model is another widely used method for the risk assessment of airborne pathogens. The Wells–Riley equation was originally developed for the epidemiological study on a measles outbreak.143 The Wells–Riley model directly combines the stable state from the box model and the one-hit exponential dose–response model, e.g., the dose–response model developed for SARS-CoV.110 The comparison between Wells–Riley model and the CFD risk assessment has been conducted for indoor spaces.144Quantitative infection risk assessment based on detailed exposure evaluation using computational fluid dynamics methods was also conducted. It was estimated that the infection risk was from 10The various risk levels obtained in different studies were mainly caused by the diverse adopted viral shedding rates and dose–response relations, which suggests that these two factors are the dominant contributors to the uncertainties of modelling-based risk assessment.3.3.2145 In the SEIR model, the total  population is categorized into four classes, i.e., susceptible, exposed, infected-infectious, and recovered, and the number of population in each class evolves with time. The SEIR models have been adopted to evaluate the effectiveness of different intervention measures.146,147 The probability of disease transmission per contact is a key parameter in the SEIR model. The exposure assessment and dose–response relations could be applied to better quantify the probability and improve the performance of SEIR models for community risk assessment.The susceptible-exposed-infectious-recovered (SEIR) model has been widely utilized to investigate the community infection risk.Modeling-based indirect risk assessment of airborne pathogen transmission has some apparent disadvantages that need to be considered. One drawback is that it relies heavily on assumptions, which may not always accurately reflect the real-world conditions. The models are based on limited data and may not capture all the complexities of the transmission dynamics. Another disadvantage is that the models may not account for individual variability in susceptibility and behavior, which can have a significant impact on the transmission risk.The direct risk exposure quantification approaches by leveraging on-site biosensing device is an essential and promising tool for infection risk assessment because it allows for a more accurate and precise estimation of the likelihood of pathogen transmission and infection. This method involves quantifying the number of contagious pathogens present in a given ambient environment as well as the duration and intensity of exposure. By taking into account these factors, on-site airborne pathogen detection and direct bioaerosol exposure quantification method can provide a more realistic assessment for airborne pathogen transmission than other methods that rely solely on qualitative observations or assumptions.In the following three sections, we will focus on introducing the emerging on-site bioaerosols sensing technologies, which will allow for targeted and more effective interventions to reduce the risk of airborne pathogen transmission.4148 Bioaerosols larger than 5 μm are mainly deposited in the nasopharyngeal region, while smaller aerosols could penetrate deeper into the lungs and deposit in the bronchiolar and alveolar regions.149 Moreover, Brownian motion further facilitates the diffusion of nanoscale aerosols or single virus particles (ca. 100 nm) and leads to high infectivity in the lower respiratory tract. Therefore, the aerosol sampling devices should have high collection efficiency in a wide size range to avoid the underestimation of airborne infection risk. In addition, there may be structural and biomolecular damages of airborne pathogens during the sampling process. This may lead to inappropriate results in subsequent quantitative analysis. In this section, we will fully evaluate these aspects in rapid airborne pathogen sampling and demonstrate recent advances that benefit effective on-site airborne pathogen quantification.On-site bioaerosols detection with high spatiotemporal resolution can be a more direct approach to characterize the transmission of airborne pathogens. Notably, an efficient and fast airborne pathogen sampling strategy is the first and crucial step. The bioaerosols should be collected at the interface between the exposure medium (indoor air) and the individual (the breathing zone). The aerosol properties, such as the aerodynamic size and pathogen decay, as well as the impact of specific sampling approaches including loss and damages should be fully considered. For instance, a very wide size range (0–100 μm) of pathogen-laden aerosols can be inhaled and deposit onto different sites of the respiratory tract, as shown in Ideally, the criteria for selecting an optimal aerosol sampling system for on-site bioaerosol quantification depends on following factors: (1) be fast and highly efficient, (2) cause less damage to the recognition biomolecules (proteins or nucleic acids), and (3) be easy-to-integrate for rapid subsequent bioanalytical testing. We summarize the existing mainstream strategies of pathogen-laden aerosols sampling and provide insights into the advantages, potentials, and critical issues of different technical routines. Regarding the three main technical requirements of the “ideal bioaerosol-sampler”, we also review some emerging technologies proposed in recent years. Meanwhile, the potential strategies to eliminate the pathogen loss and damage during the sampling process are also discussed to further benefit the future development.4.14.1.111,150–152 Particularly, the airflow rate is one of the important characteristics for on-site pathogen-laden aerosol collection. High airflow rate combined with superior sampling efficiency could load more airborne viruses onto the filter, therefore facilitating swift biosensing tests to provide detection results with high spatiotemporal resolution for risk assessment and determine the potential airborne pathogen transmission route.20,150 By elevating the sampling flow to 150 L min−1, the positive detection rate of airborne SARS-CoV-2 in the inpatient ward environment was significantly improved (72%), while the lower sampling flow rate (50 L min−1) resulted in more negative detection results in similar hospital air settings. As a robust filtration matrix, PTFE filter is resistant to longer sampling periods and higher flow rate compared to fragile filters.153 However, the use of filtration methods for bioaerosols collection could lead to dehydration, which deactivates the collected pathogens and potentially damages the functional biomolecular structure. Boosting the flow rate could also seriously exacerbate this disruption and significantly affect pathogen viability and integrity. Therefore, the losses need to be considered when analyzing the bioaerosol content on the filter. For instance, pathogen integrity-based assays, which rely on the structure or functionality of an intact virus or bacteria, are not suitable for analyzing filter-collected samples. In contrast, integrity does not have to be a predominant focus for biomolecular-based approaches, such as viral RNA or bacteria DNA quantification, as these analytes can still be detected even after the loss of their viability.Filtration is one of the most effective, robust, and commonly used strategies for airborne pathogens collection . Through34 The water-dissolvable filter material can mitigate this inconvenience and achieve rapid liquid sample preparation for subsequent biochemical analysis. As a prominent candidate, dissolvable gelatin filter can significantly reduce bioaerosol damage and losses by exempting the sample elution and extraction processes. The physical collection efficiency of a gelatin filter for submicron bioaerosols is given in ca. within 2 min) after bioaerosol sampling.40The elution of airborne virus from the filter is another key factor that may impact the accuracy of subsequent quantitative detection. Significant losses in bioaerosol elution and extraction from filter substrates may result in a false negative detection result.154 For instance, high RH can damage the water-dissolvable filters, while high temperatures can accelerate the melting of the filter structure. One study demonstrated that a stable high collection efficiency of gelatin filter was only sustained for 9 min at 2 L min−1 flow rate before filter degradation.155A miniaturized filtration sampler equipped with a water-dissolvable filter could be a straightforward strategy to collect airborne pathogens on-site. Currently, the filtration-based sampling approaches were commonly incorporated with a nucleic acid detection technique, and its preservation performance for other functional biomolecules (antigenic epitopes) still needs further validation. In addition, the durability and robustness of water-soluble filters for airborne pathogen collection is a concern as environmental factors can directly impact the filtration efficiency.4.1.2156 The impaction efficiency for aerosols heavily depends on the physical properties of airborne particles  as well as the velocity of driving airflow. One of the main advantages of using impactor-based sampler is its capability to separately collect bioaerosols with different size fractions.38 By tuning the inlet nozzle diameter, a series of sampling stages in a cascade impactor could be connected in sequence, and each plate has a gradually decreased nozzle dimension. Airflow passing through a smaller nozzle has a higher velocity, therefore allowing smaller aerosol particles to be collected. Recently, the cascade impactor was utilized to investigate the SARS-CoV-2 virus concentrations and distributions in different size-groups, e.g., large (>10 μm), coarse (2.5–10 μm), and fine (<2.5 μm) aerosols.11,38,157 One comparative study also demonstrated a low SARS-CoV-2 infectivity loss over half-hour sampling using a cascade impactor, which indicated the potential for effectively collecting intact and viable pathogens.38 Due to the nature of inertial impaction, the sampling efficiency is typically high for large and dense bioaerosols but insufficient for nanoscale bioaerosols. Therefore, impactor-based sampling approaches are mainly used for collecting aerosols larger than 0.5 μm.Based on the inertia effect, pathogen-laden aerosols can also be collected by an impactor. When the carrier airflow is sharply curved, the aerosols cannot follow the air streamline but travel continuously in the original direction . The imp158 For instance, extremely small nozzle and high flow velocity could induce sonic airflow and shear stress, thereby destroying the pathogens. When utilizing a cascade impactor, there are also considerable target losses in the bioaerosol elution and transfer at each sample stage.As mentioned in Section 2, nanoscale bioaerosols have a long suspension lifetime. Due to its stronger Brownian motion and potential to penetrate into the lower respiratory tract, smaller aerosols are a non-negligible part of on-site airborne pathogen detection and risk assessment. Although the collection efficiency toward small-sized bioaerosols can be theoretically improved by reducing the nozzle inlet diameter, this approach also brings associated problems.4.1.3159–162 For instance, an increased cyclone body height could elevate the aerosols residence time and improve the collection efficiency.159 Since this method also relies on the inertia of target aerosols, cyclone is also more efficient to collect larger particles with higher densities.160 Though raising the flow velocity can increase the overall performance, the collection efficiency of submicron bioaerosols is still poor.162 Cyclone-based samplers have been widely employed in the collection of airborne pathogens due to their portability and simplicity of liquid elution and transfer. A wearable multi-stage cyclone bioaerosol sampler has been used for the collection of airborne viruses in the patient wards.34 Bioaerosols with diameter of 1–4 μm and >4 μm can also be simultaneously collected using a commercially available cyclone sampler , and the pathogen sequences have been successfully collected and detected in both size-groups.163 A high volume (400 L min−1) cyclone sampler was also integrated with a medical robot for the smart sampling of airborne pathogens.164 This cyclone-based sampler demonstrated a physical collection efficiency of >90% for bioaerosols larger than 1 μm, and the cutoff size of sampling d50 was determined to be 0.58 μm.Centrifugal force is utilized in cyclone-based sample techniques to deflect inertial aerosols from the airflow and achieve effective airborne pathogens collection . Ambient163 Therefore, in addition to the impact of external environmental factors, negative test results may potentially be caused by the low sampling efficiency of airborne pathogens.165Despite the advantages of miniaturization and ease-of-integration, the low collection efficiency for submicron bioaerosols may lead to a significant underestimation of the quantification of airborne pathogens, especially the viral particles and submicron-scale bacteria. A previous study has indicated that more than half of the viruses cannot be efficiently collected by cyclone-based samplers.4.1.4166,167 The incoming aerosols are commonly charged by a corona discharge in case they carry insufficient electric charge for efficient sampling. The advantages of the electrostatic sampling approaches for airborne pathogen collection include easy-to-miniaturize for portable applications (<1.0 kg and battery powered), high collection efficiencies (>90% for aerosols larger than 1 μm), wide volumetric flowrates (1 to 1000 L min−1), wide sampling size range (0.5–200 μm), and low pressure drops during operation.166,168–170 Ultra-high collection efficiencies (99.3–99.8%) can be achieved when using a low flow rate (1.2 L min−1) at −10 kV applied voltage  and reactive nitrogen species (RNS) upon exposure to a strong electrostatic field, such as H2O2via lipid oxidation-derived radicals and 1O2via direct protein peroxidation.17,174 It was found that more than 90% membrane proteins of airborne virus lose antigenicity after 10 min of electrostatic sampling when using an applied voltage of −5 kV and a 1.2 L min−1 airflow rate  from patient exhalation.176It is worth noting that airborne pathogens may suffer a considerable loss in viability after the electrostatic-based collection, as illustrated in low rate .17 The o4.2Biomolecule recognition, such as protein-epitope interaction and nucleic acid hybridization, are crucial for airborne pathogen detection. However, these recognitions can be affected by changes in the environment, such as temperature, pH, and humidity. Dry conditions, in particular, can exert a significant impact on biomolecules, leading to alterations to their structural conformation and functional properties. Therefore, liquid-phase sampling approaches, which directly achieve aerosol-to-hydrosol collection, are often favored for the on-site detection of airborne pathogens.4.2.1There are two obvious problems with solid-phase sampling methods for on-site airborne pathogen detection . FirstlyTo address these shortcomings, a convenient approach is to carry out aerosol-to-hydrosol modification of the conventional solid phase sampling techniques including impactor, cyclone, and electrostatics samplers. The liquid collection medium, instead of a solid deposition interface, helps maintain the viability and integrity of the virus. Direct collection and enrichment from aerosols to hydrosols for on-site airborne pathogen detection might enhance the preservation and make it easier for the following downstream bioanalytical system to accomplish near-real-time detection. This direct aerosol-to-hydrosol sampling method not only eliminates the time consumption for sample preparation such as bioaerosol elution and transfer but also avoids target losses within this pre-treatment process.177 For instance, a liquid transfer stage has been utilized to replace the impaction receiver in the aerosol-to-hydrosol impactor, allowing bioaerosols to be directly collected from the airflow into the liquid flow.178 This virtual impactor was used to sample airborne pathogens in the hospital environments, and the bioaerosols can be directly collected into the sterile PBS buffer solution for downstream analysis.179The aerosol-to-hydrosol sampling approaches such as the virtual impactor with a liquid transfer stage, wetted-wall cyclone concentrator, and liquid-phase electrostatic sampler have been adapted to collect airborne pathogens in recent years.164 In one pioneering study, a wetted-wall cyclone was deployed to collect SARS-CoV-2 aerosols in hospital wards and successfully facilitated the downstream tests to estimate the aerodynamic distribution and transmission distance (∼4 m) of virus-laden aerosols.180Wetted-wall cyclone concentrators utilize water spray to wet the internal surface of the conical-shaped cyclone body and directly collect aerosols into a small amount of aspirated water buffer.168 Apart from the discharge electrode, a thin layer of pathogen lysis buffer was utilized as a collection electrode within the system. The collection efficiency can be optimized to reach about 88% for human HCoV-229E coronaviruses. In another liquid electrostatic sampling system, approximately 80% of SAR-CoV-2 virus-laden aerosols ranging from 100 nm to 5.0 μm can be collected with an airflow rate of 40 L min−1.172 To further improve the stability and integrity of the collected airborne pathogens, the chemical compositions of the sampling liquid could be further optimized. For instance, protective agents like ascorbic acid (AA), sodium azide, and uric acid can be added to the PBS sampling liquid reservoir to mitigate the inevitably damage caused by the generated ROS and RNS from electrostatic precipitation  was proposed to collect various types of airborne pathogens including human coronavirus 229E, influenza A virus subtypes H1N1 and H3N2, and bacteriophage MS2.pitation .181 Thes182 For downstream biosensing, a recent study has demonstrated that additive glycerol promoted the detection of African swine fever virus (ASFV) and SARS-CoV-2 with one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a method and a smartphone-equipped device.183 During the initial phase of the RPA reaction, the viscosity of glycerol led to the phase separation of the RPA and CRISPR/Cas12a system, which increased the detection efficiency. Nevertheless, the detection effectiveness in different liquid environments containing glycerol may require further validation for specific biosensing technologies, including the selective recognition between different biomolecules.The conventional approach for aerosol-to-hydrosol sampling involves the utilization of water or other liquids with low viscosity as the medium for collection. Hence, the evaporation issue at the “dry-to-wet” interface can also have an impact on the performance of sampling. This issue not only restricts the overall sampling duration to shorter time periods but also impacts the efficiency of sampling and the viability of pathogens. The non-evaporating and higher viscosity liquids, such as glycerol and heavy white mineral oil, have been proposed to sample airborne bacteria and fungi for several hours.4.2.2184,185 In an liquid impingement system, the airborne particles are transported and accelerated to high velocity through a circular or rectangular nozzle and then expected to impact into a collection liquid through inertia. By leveraging the aerosol-to-hydrosol approach, liquid impinger-based samplers could improve the viability and retention of the airborne pathogens. Meanwhile, the high-speed airstream creates a high degree of turbulence and stimulates the formation of bubbles inside the sampling liquid, which could increase the collection efficiency for tiny bioaerosols , while a high efficiency of >90% was observed at 10 nm due to the enhanced Brownian diffusion inside the swirling sampler.155,188,189 Additionally, the inlet design of all-glass impinger (AGI) and BioSampler, which mimic the human upper respiratory tract, could lead to a low inlet efficiency of about 80% for aerosols larger than 5 μm.190 Therefore, these effects, including the size-dependent inlet efficiency and sampling efficiency, must be taken into account when conducting the on-site sampling-to-biosensing–based infection risk assessment. A more precise estimate of the airborne pathogen concentration can be obtained by considering the size-fractionated airborne pathogen distribution.As a representative of the swirling aerosol collector, the commercial SKC BioSampler provides an improved performance on reducing the reaerosolization.4.2.3191 The wet-scrubber-based sampling approach collects aerosols via direct scavenging with liquid droplets. This approach combines multiple sampling mechanisms including the turbulent diffusion, inertial impaction, interception, and gravitation to achieve the effective collection of airborne particles.192,193 During the sampling, bioaerosols collide and merge with liquid droplets through coalescence. In conventional industrial wet-scrubbers, liquid droplets generated by pressurized nozzles cannot efficiently collect airborne particles with diameter less than several micrometers. Additionally, the collection liquid volume is generally large, and the airborne virus particle cannot be beneficially concentrated for downstream analysis. To tackle these issues, many novel strategies have been applied to modify the wet scrubbing-based collector, including twin fluid atomization, droplet charging, high-pressure spray, and ultrasonic microdroplet generation.194–196 Recently, a portable and battery powered wet-scrubber device was developed for collecting airborne pathogens.191 This device generated a mist of 4 μm liquid droplets in a non-linear flowing path. By combining the impaction, diffusion, interception, centrifugation, and liquid scavenging effect, this miniaturized aerosol-to-hydrosol scrubber device demonstrated an improved sampling efficiency and viable pathogens preservation for rapid downstream analysis.An alternative aerosol-to-hydrosol approach to collect airborne pathogens is based on the liquid droplet sprays and wet scrubbers.Despite an improved bioaerosol sampling performance, the wet scrubber still demonstrated a lower collection efficiency for nanoscale bioaerosols compared with other high-efficiency sampling approaches. To prevent false-negative results and underestimation of the transmission risk of airborne pathogens, practical on-site pathogen-laden aerosol quantification still needs a lengthy sampling period and improved efficiency for small bioaerosols.4.317,177,197–202 Compared to the conventional sampling approaches, microfluidic chips with miniaturized physical structural design have shown the advantages of low cost, fast response, automation, and compatibility for fast on-site bioanalysis.203 As shown in 197–200,202,204With the rapid developments and advancements in microfabrication technologies, microfluidic-based bioaerosol samplers have offered a promising avenue for swift airborne pathogens collection and rapid subsequent detection.199 In order to ensure stable micro-bubble generation and high collection efficiency, the inner surface of microchannel and micro-pillar array was coated with hydrophobic Teflon layer. The proposed micro-impinger was able to collect and concentrate the aerosol particles into a much smaller solution volume (250 μL) with a gentle airflow rate (10–20 mL min−1) when compared to the conventional impingement collector. For bioaerosols larger than 0.5 μm, the micro-impinger achieved a satisfactory collection efficiency of >95%. However, further investigation is still needed to fully understand the sampling performance toward nanoscale bioaerosols, such as a single airborne virus particle, as shown in For instance, based on the principle of Greenburg–Smith impingement sampler, a miniaturized micro-bubbler has been developed for gas-particle mixing, bubble release, and aerosol-to-hydrosol sampling .199 In o200,204 Instead of direct solid-phase impaction on the channel, a two-phase microfluidic system, which combined the sampling airflow and sheath fluid, was developed to directly transfer the airborne pathogens into liquid in a curved microchannel . Moreover, the inertial and centrifugal forces can be alternatively used for continuous aerosol size separation, as shown in e.g., <0.5 μm, 1–2 μm, and >3 μm).205 Despite this approach, it does not achieve a high enrichment efficiency for bioaerosols by itself, it enables downstream sampling and bioanalytical devices to acquire the size distribution of virus-laden aerosols, much like the traditional multi-stage impactors. Apart from inertia-based sampling, external deflection forces such as electrostatic trapping forces have be utilized to precipitate or manipulate airborne particles in a microfluidic system.170,202In addition, airborne particles can also be collected in microfluidics by leveraging the technique of centrifugation.ochannel .200 This206 For instance, the herringbone grooves located on the inner channel surface can induce the micro-vortex and enhance the turbulent deposition of bioaerosols at the inner surface of the microfluidic channel  during sampling, and Vliquidflow is the volumetric liquid flow rate (in L min−1) of the collection medium, or instead refers to the volume of static collection medium per unit time . The ESEC values for some of the microfluidic-based and conventional aerosol-to-hydrosol sampling approaches are given in Compared with conventional pathogen-laden aerosol sampling approaches, microfluidic-based samplers provide a number of exceptional advantages. Miniaturized microfluidic samplers obviously outperformed conventional samplers in terms of integration level, low power consumption, and post-processing simplicity for on-site airborne pathogens quantification and real-time risk assessment. However, there are also obvious issues and challenges that require immediate attention. For instance, the blockage of microfluidic channels can be a potential issue that affects the performance and reliability of airborne pathogen and bioaerosols sampling. Additionally, there is still room for improvement in the effectiveness of collecting nanoscale bioaerosols, particularly for the majority of airborne viruses with diameters as small as 100 nm. Although the microfluidic-based bioaerosol sample may enrich the bioaerosols into a smaller volume of solution, the airflow rate is also noticeably decreased compared to traditional approaches. Therefore, it can be beneficial to use the effective sampling enrichment capacity (ESEC) to assess the overall effectiveness of the sampling system, which is given by−1 may not be practical for real-time and rapid pathogen quantification. Hence, it is crucial to conduct further validation studies to assess the applicability of a microfluidic-based sampling system regarding various detection scenarios and types of airborne pathogens.The concentrations of fully diluted airborne pathogens are typically at an extremely low level compared to patient specimens. For microfluidic-based sampling devices, which can handle only a limited volume of air, it is difficult to collect sufficient number of pathogens for downstream analysis even if a high ESEC value is achieved. For instance, a microfluidic-based sampling system with a limited air flowrate of 2 mL min4.439,207–209 Condensation growth approach, as shown in 209,210 For instance, the condensation growth approach can directly improve the physical collection efficiency of swirling bioaerosol sampler from 10% to 99% for 100 nm bioaerosols.209 The viability preservation of airborne MS2 bacteriophage was also improved by 22 times compared to the bare commercialized BioSampler. The condensation-based bioaerosols sampler have also been deployed for collecting airborne virus in different patient environments.211 Taking advantage of the gentle aerosol-to-hydrosol sampling process and condensation-induced high sampling efficiency, viable viruses in concentrations ranging from 6 to 74 TCID50 units per L have been successfully identified in patient wards.211Based on the physical properties of the pathogen-laden aerosol (see Section 2.3), a number of airborne pathogens, especially virus and small size bacteria, can be carried by submicron aerosols. Despite having a low mass content, nanoscale bioaerosols may be more prevalent and cause a non-negligible risk of infection because of their capacity to traverse large distances and deposit themselves directly on the low respiratory tract. Consequently, it is essential to increase the sampling effectiveness of collecting submicron-scale bioaerosols. This is particularly true with microfluidic-based samplers, which have a poor collection efficiency for bioaerosols smaller than 0.5 μm. Condensation growth-based approach, also known as the hygroscopic growth, and mixing-type bioaerosols amplification unit (mBAU), is one of the most common and effective strategies to improve the physical collection efficiency of submicron-scale aerosols.212,213 Compared to the conventional bench-top devices, hygroscopic systems were not only far more compact, affordable, and power-efficient but also demonstrated remarkable performance in facilitating the optical detection of bioaerosols down to 10 nm. By integrating with microfluidic-based samplers, as reviewed in Section 4.3, these micro-condensation aerosol sampling systems may be able to provide excellent sampling efficiencies over a broad aerodynamic size range from 10 nm to 100 μm, thereby offering solid support for on-site airborne pathogens detection and accurate risk assessment.In addition, the condensation growth device has also been miniaturized into a portable microfluidic system by harnessing 3D printing, photolithography, and passive cooling techniques.4.5214,215 Additionally, in some public areas, the concentrations of a particular type of pathogen floating in the air may be quite low. However, prolonged exposure in such environments may still result in a cumulative intake of pathogens or bioaerosols at a minimum infectious dose. Since the majority of biosensing systems for pathogens or bioaerosols detection possess a ‘non-zero’ detection threshold , negative results might not completely rule out the potential of infection and health impact. Target selective-enrichment and preconcentration prior to quantitative bioanalysis may greatly benefit on-site biosensing detections and boost the accuracy of on-site risk assessment. In this section, pre-treatment strategies including impurity removal, selective target enrichment, and effective to-be-tested sample delivery for downstream biosensing are reviewed and discussed.The obtained aerosol samples may include a wide range of complicated chemical and biological components dispersed in the air. During the bioaerosols treatment, these compounds may impact the integrity of the collected airborne pathogens and make it challenging to identify them later using bioanalytical methods. For instance, after being enriched in the sampler, metal ions and ROS in the air may have an impact on the biomolecular structure and hasten the breakdown of whole pathogens or its particular recognition sites (such as proteins and nucleic acids). This might lead to inaccurately low airborne pathogen concentration readings or false negative findings.198,216,217 Considering the necessity of fast processing for on-site bioaerosols detection, we place an emphasis on rapid enrichment systems that can be miniaturized rather than large and resource-intensive bench-top equipment. Recently, a 3D porous microfluidic system with a robust array of aligned carbon nanotubes (CNTs) was proposed for virus purification in field samples , the nitrogen-doped CNT array can be further patterned into a herringbone structure to enhance the mixing of the sample inside the microfluidic channel through the chaotic flow.219 In addition, the stamping method was able to pattern Fe catalytic particles with a density gradient, resulting in the formation of multiple herringbone CNT zones with different physical inter-tubular distances ranging from 22 to 720 nm. The integration of CNT array zones with varying inter-tubular distances can be used not only to enrich a broader spectrum of airborne particles but also to purify bioaerosols of different sizes on the same device and facilitate size-resolved pathogen biosensing.Based on the physicochemical characteristics of airborne pathogens, various technologies that leverage physical separation and affinity-based extraction methods can be applied to purify the target bioaerosols. samples .218 Thise.g., solid-phase extraction and hydrogel protein purification) target specific pathogen recognition sites, such as nucleic acids or proteins.220–222 Recently, an affinity-based microfluidic centrifugal device was developed for enriching viral RNA into a detection-ready sample.220 This centrifugal microfluidic device leveraged the magnetic virus-affinity particles to isolate intact pathogen sequences through multiple lab-on-chip processes including magnetic manipulation, affinity binding, flushing, and RNA elution. This integrated microdevice can rapidly remove complex matrices from the field aerosol-to-hydrosol samples within 15 min. Moreover, chemically modified polymers, such as hydrogels with affinity baits, can be used to purify and enrich pathogens via interactions with target proteins.221,223 Recently, affinity-based hydrogel nanoparticles (Nanotrap) have been used to swiftly enrich SARS-CoV-2 by binding with viral spike proteins.224 The entire preparatory procedure, including virus enrichment, enzymatic RNA extraction, and elution, required approximately 10 min.In contrast to physical separation, affinity-based enrichment techniques  or trigger unexpected binding to biosensors . Various approaches have been proposed recently to improve the biosensing accuracy, including the use of blocking agents, sample pretreatment, and novel intrinsic biosensor design .18,228–230 Meanwhile, a novel biosensor design could also enable on-site pathogen sensing system to work in different harsh environments.In a manner different from existing clinical diagnosis approaches, on-site bioaerosol sensors need to be more sensitive, accurate, robust, and swift for versatile, automated, and connectable sensing applications. To further clarify the research priorities for the on-site airborne pathogen biosensor development, we propose a set of “18,19,231,232 In addition, biosensors should be versatile and readily adaptable to detect various types of airborne pathogens and variants, given the rapid mutation and emergence of novel pathogens. Last but not least, automated and connectable sensor network could allow for measurement of multilevel  exposures, i.e., with a high degree of spatiotemporal resolution. On-site airborne pathogens sensing services can be connected to the central network and provide real-time accessible data to facilitate evidence-based interventions of airborne pathogen transmission.Swiftness is another important trait to allow exposure measurement with high spatiotemporal resolution and early-stage warnings. Currently, numerous biosensors can detect trace quantities of pathogens within 10 min.SARSVAC” criteria could further clarify the development and research priorities of airborne pathogen biosensing technologies. In the sections that follow, we will review various on-site airborne pathogen detection strategies, highlighting the recent advancements and prospective challenges.The proposed “5.1Streptococcus, and Legionnaire's disease. Viruses are even smaller infectious agents that can cause a range of ailments, from the common cold to more severe diseases such as COVID-19, chickenpox, monkeypox, and measles.233 For instance, coronaviruses are enveloped single-strand positive-sense RNA viruses with an average diameter of about 100 nm. As shown in 234,235 Fungal aerosols, which include yeasts and spores, are the third group of airborne pathogens.50 Some types of fungi can also cause respiratory infections such as aspergillosis and histoplasmosis. For specific pathogens or general bioaerosols, there are mainly two groups of specific recognition targets for on-site and rapid detection: nucleic acids (DNA or RNA sequences) and antigenic epitopes (antigen proteins).Airborne pathogens may consist of bacteria, viruses, fungi, or other microorganisms, as shown in 5.1.1Pathogens contain either DNA or RNA as their genetic material. DNA is the genetic material found in most microorganisms, including bacteria and viruses like adenovirus, whereas RNA is found in certain viruses such as influenza virus, SARS-CoV, and SARS-CoV-2. From a bioanalytical point of view, these unique sequences, as shown in 236 Similarly, the gp43, p27, and ITS genes are all genetic biomarkers for fungal identification and classification. ITS sequence located between the 18S and 28S region is highly variable and evolves rapidly, making it useful for identifying and differentiating between fungal species.23716s rDNA is a conserved gene sequence found in all bacteria and extensively used for the identification and classification of bacteria. The general 16s rDNA sensing techniques involves the amplification of the 16s rDNA gene from the bacterial sequence, followed by the sequencing of the amplified product to identify the bacterial species present in the bioaerosols sample. Because of the universality of this method for bacterial detection, the recognition site of 16s rDNA has been very versatile in both clinical and environmental sensing applications.238 In clinical diagnosis, nucleic acid detection, e.g., polymerize chain reaction (PCR), is currently considered as the gold standard thanks to its well-characterized sensitivity and accuracy. For instance, genomic probing sites located in N gene (encoding nucleocapsid protein), Orf1ab gene (encoding open read frame 1ab protein), E gene (encoding envelope protein), RdRp gene (encoding RNA dependent RNA polymerase), and S gene (encoding spike protein) shown in 34,239 Integrated bioaerosol sampling strategies, such as the inertial impactor embedded in a micromachined silicon chip, have been recently proposed for facilitating point-of-care RT-qPCR detection of the airborne SARS-CoV-2 viruses.240 However, several characteristics of PCR tests including being resource-intensive, cumbersome, and time-consuming hampers their applications in real-time airborne virus detection and on-site virus transmission monitoring. Numerous high-performance, quick, and practical nucleic acid detection technologies have been suggested and developed recently.241,242 As an prominent gene-editing technology, clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR/Cas)-based assays have been proposed for the sensitive and swift recognition and detection of pathogen genes.225,243–245 In the CRISPR/Cas-based biosensors, the cleavage activity of the nuclease-inactive ribonucleoprotein complex, which contains Cas-protein and crRNA, can be selectively activated by binding to the complementary sequence of the pathogen target.246 The activated complex can be used to indiscriminately cleave any surrounding sequence and generate a sensible signal. For instance, the CRISPR-Cas13a assay was developed for the direct detection of SARS-CoV-2 RNA with a superb sensitivity by 100 copies per μL and a swift turnaround of 30 min.225 Although this sensing approach was initially designed for clinical diagnosis, its swift and sensitive characteristics meet the criteria for on-site pathogen detection. Additionally, this CRISPR-based biosensing assay can be integrated with a mobile phone-based readout device to fulfill the criteria of prospective sensor networking and connectable IoT. To date, the CRISPR/Cas approaches have not been directly applied to detect on-site collected aerosol samples. The sensing performances such as recognition accuracy and stability need to be further verified when the assay reagents are directly exposed to diverse aerosol compounds from the environment.Due to the diversity of viral families, it is difficult to find a universal genetic marker for detecting all types of viruses. For DNA viruses such as adenoviruses and smallpox virus, one conserved genetic region is known as the DNA polymerase gene, which encodes for the enzyme responsible for DNA replication. Similarly, the RdRp (RNA-dependent RNA polymerase) gene, which may be utilized as a target for identifying airborne viruses, is present in a variety of RNA viruses, including coronaviruses and influenza viruses. Coronaviruses such as SAR-CoV and SARS-CoV-2 possess a positive-sense RNA sequence with 26 to 32 kilo-bases in length.A major disadvantage of nucleic acid detection approaches for usage in on-site airborne virus detection is the demand for additional sample pretreatment procedures, including pathogen lysis, release of inner nucleic acids, and removal or inactivation of NA-degrading enzymes. An integrated microfluidic system, as explained in Section 4.5, is more appealing for the sampling, pretreatment, and delivery of pathogen aerosol samples for biosensing.5.1.2247–249 A fast bacteriological immunoassay and an integrated microfluidic device capable of capturing and enriching airborne microorganisms like Mycobacterium tuberculosis were also developed. In addition, aptamer or synthetic polymer receptors  can also be utilized to detect antigen.248–250The antigenic epitopes of airborne pathogens represent another group of recognition sites for quantitative biochemical analysis. KD) between the receptor and target proteins. Taking the SARS-CoV-2 spike protein as an example, the binding affinity with human angiotensin-converting enzyme 2 (ACE2) receptor is about 14.7 nM.251 In contrast, two optimized oligonucleotide aptamers selected through systematic evolution of ligands by exponential enrichment (SELEX) possess affinities of 5.8 nM and 19.5 nM.249The epitopes recognition strategy is mainly based on the binding affinity .The most outstanding advantage of epitope-based recognition strategies is their swiftness and flexibility. Generally, most of the recognition approaches by antigenic epitopes take less than 30 min, which may benefit the on-site and real-time airborne pathogen quantification. Ideally, the epitopes receptor of biosensors should be selective and specific to the designated target protein. However, the majority of protein receptors, including pathogen antibodies, are promiscuous and may bind with a variety of non-specific biomolecule.5.2SARSVAC” criteria. The potential strategies and novel designs for improving the bioaerosol sensing performance are summarized in Selective biosensing receptors targeting pathogen recognition sites require sensitive transducing elements to transform the molecular interactions into quantifiable analytical signals. These transducing elements can be a photo-responsive matrix, a nanomaterial-based electrode, or an interface of a piezoelectric microbalance. Therefore, based on the different physiochemical transducing principles, biosensors can be readily classified into several different groups, namely, electronic/electrochemical, optical, and mechanical/acoustic biosensors. Hereby, we review the novel progresses of high-performance biosensing systems for on-site airborne pathogen detection and further provide insights following the “5.2.1259 Based on the transducing signals, electrochemical biosensing approaches can be divided into categories of amperometry , conductometry , voltammetry , and potentiometry .260,261In electronic/electrochemical biosensing systems , conduct262 Additionally, electrochemical biosensors can be easily integrated with existing sensor networks due to their easy interface with conventional electronic read-out.263 Field-effect-transistors (FETs) and miniaturized potentiometric systems are the prominent instances for airborne pathogen detection.232,264 Seo et al. proposed a graphene-based FET biosensor for detecting the SARS-CoV-2 virus in different liquid media , as shown in 232 According to the preliminary biosensing results, the FET-based biosensor possessed a detection limit of 242 copies per mL through the direct recognition of the antigenic epitopes. In addition to this superior sensitivity, electrochemical biosensors also possess the advantages of easy-to-manufacture and integration. A split aptamer (SA)-based electrochemical sensor chip was recently developed for the rapid detection of adenosine triphosphate in bioaerosols with a detection limit of 10 pM, which can be used for the ubiquitous detection of total bioaerosols.265Based on the fast development of low-cost microelectronic circuits and nanofabrication technologies, electrochemical-based biosensors demonstrate advantages of high robustness, low-cost, easy miniaturization, and low system complexity.e.g., paper, polymers, and clothes).266–269 For instance, the screen-printing method could enable the mass manufacturing of electronic pathogen sensing devices.270,271 Recently, a paper-based voltametric electrochemical biosensor was developed for rapid and on-site airborne influenza virus quantification.17 This voltametric biosensor had a fair detection limit of 2.13 PFU per mL. Due to the integrability of electrochemical systems, this low-cost and paper-based biosensor has been directly incorporated with a portable electrostatic aerosol concentrator for the continuous monitoring of virus-laden aerosols. In another recent work, Xue et al. integrated the conductive nanowire-based immunosensors, a miniaturized flexible impedance circuit and a wireless communication unit into an intelligent face mask for personal infection detection.247 Through the enrichment of bioaerosols by the facemask, this electrochemical biosensor could directly detect airborne coronavirus at a concentration of 0.35 PFU per L in air. By integration with aforementioned airborne pathogen collection, enrichment, and pretreatment units, as shown in Additionally, the transducing electrodes of electrochemical biosensors can be easily integrated onto different flexible or low-cost substrates  in a microfluidic system can be further developed into a multi-functional electrochemical system, in which the airborne pathogens can be simultaneously enriched  and in situ detected.218Although electrochemical biosensors offer a promising avenue for on-site airborne pathogen detection, their capability to detect trace amounts of specific target in complex aerosol matrix remains a major concern.5.2.2276,277 On the basis of optical modulating and transducing configurations, photonic biosensors can be divided into numerous classes, including optical resonance, dispersion, reflection, refraction, absorption, Raman scattering, and fluorescence. Utilizing nanomaterial-enhanced light-matter interactions, nanophotonic devices have demonstrated superior biochemical analysis performance for transducing trace amount of analytes.276 Among them, plasmonic enhanced light–matter interaction has proven to be an invaluable technology for the highly sensitive detection of pathogens such as bacterial and viral particles.44,278Photonic pathogen sensors present another promising approach for highly sensitive coronavirus detection .276,277 279 Numerous LSPR-based biosensing systems, modulated via reflection, absorption, or phase, have been employed for detecting trace amounts of airborne pathogens.18,44,280–282 Moitra et al. proposed to use colloidal plasmonic nanoparticles for colorimetric viral gene quantification through rapid “naked-eye” detection.282 The same research group further integrated this plasmonic enhanced light–matter interaction technique into a hyperspectral sensor, which exhibited the hallmark of ultrahigh sensitivity at the yoctomole level and rapid turnaround time of a few seconds.281Localized surface plasmon resonance (LSPR) indicated that photon-induced collective electrons oscillation resulted from the nanomaterial-enhanced light–matter interaction.283 A prominent example is using thermoplasmonics, an enhanced photothermal effect to regulate the thermodynamic interactions between target genes and oligonucleotide receptors, as shown in 44 The proposed thermoplasmonic heating was able to increase the local temperature for nucleic acids recognition and facilitate the elimination of interference from nonspecific objects and spurious pathogenic genes.44 The same research group also developed a plasmonic biosensor for total bioaerosol detection.284 By leveraging the succinimidyl-ester surface functionalization, total bioaerosols can be quantitative detected with high sensitivity down to 0.5119 cells per mL.In addition to improving the operability and sensitivity, smart optical sensors designs may significantly improve the biosensing reliability and robustness.285 Raman spectroscopic strategies are able to provide a unique spectral fingerprint generated by the interaction of light with the molecular vibrations of the target airborne pathogens.286 One research employed angiotensin converting enzyme 2 receptor-functionalized SERS biosensors to quickly detect viral spike proteins with a turnaround time of less than 5 min and a low LoD of 80 copies per mL.287 Additionally, the machine-learning algorithm was employed to improve the discrimination capability for identifying the pathogen-related SERS fingerprint in the complex liquid system. Choi et al. integrated an optofluidic SERS platform with a microfluidic-based aerosol-to-hydrosol impingement aerosol sampler and demonstrated the capability of continuous bioaerosols monitoring.286Nanomaterial-based surface-enhanced Raman spectroscopy (SERS) is an alternative optical route to achieve highly selective and sensitive airborne species detection.14,16,255 Lee et al. described an integrated sampling-to-monitoring system for the on-site and real-time detection of airborne pathogens.14 The antibody-conjugated up-conversion nanoprobes (UCNPs) were able to specifically bind to the collected airborne pathogens in a paper-based system, and the stimulated UCNPs photoluminescent emission could be used for on-site and rapid virus quantification with LoD down to 104.294 EID50 (50% egg infective dose)/m3. In addition, spark-induced plasma spectroscopy was also recently reported to achieve on-site and real-time detection of airborne viruses.16Recently, optical biosensing assays based on photon emission have also been utilized for airborne virus quantification and risk assessment.288 One of these products utilized a cell-based and bioluminescent-mediated biosensing technology with membrane-bound pathogen antibodies and expressed bioluminescent protein collaborated to accomplish cellular analysis and notification of antigen risks and yields (CANARY).255 In addition to these established optical biosensing approaches, advanced photonic techniques such as chiral plasmonics, dielectric nano-resonators, and surface enhanced infrared absorption spectroscopy (SEIRA) could also be potentially utilized for highly sensitive on-site airborne pathogens monitoring.289There are also commercially available optical instruments for the real-time and on-site detection of airborne pathogens.SARSVAC” criteria and achieve on-site risk assessment, the integrability and compatibility of optical biosensing devices should be further improved. For instance, 3D printed micro-photonic units could facilitate the miniaturization of bulky optical sensing systems. Moreover, optofluidics can be integrated with the micro-sampler units  with surface-functionalized bio-recognizing receptors  exemplify a revolutionary technology for rapid and sensitive biochemical analysis.296The third major class of biosensors for airborne pathogen detection is micromechanical transducer, as shown in et al. proposed a microcantilever-based bio-MEMS system for the rapid detection (<5 min) of viral spike and nucleocapsid proteins  can be integrated into the micromechanical system to further improve the biosensing performance.297 In this novel mechanical bioaerosol sensing system, a microelectronic FET unit was embedded as the base of the micromechanical cantilever to directly read the deflections caused by the biomolecular binding.Recently, Agarwal proteins . By moni298 In this miniaturized system, the resonating Si cantilever was parallelly employed as the electrostatic sampling electrode and the mechanical sensing matrix. By validating with 100 nm airborne particles, the sensor exhibited a mass sensitivity of 36.5 Hz ng−1 after 15 min aerosol sampling. By functionalizing specific protein receptors or complementary nucleic acid sequences on the micromechanical sensing substrate, the micromechanical system may be used as an integrated, rapid, and cost-effective system for on-site pathogen aerosol detection. Moreover, the miniaturized sensing volume and novel functionalization strategies also enabled multiplexed analysis with microelectronic biosensing system.299 Based on multichannel detection on the same platform, different pathogen units can be detected in parallel so as to eliminate the impact of non-specific binding events and provide more accurate bioaerosols analysis results. By supplying at least one reference channel, it is also possible to eliminate the background noise induced by the complex airborne components.300To achieve on-site aerosol detection, a sampling unit, such as an electrostatic precipitator, has been further integrated with the micromechanical sensing system.300 In addition, mechanical biosensing devices typically require additional signal reading units, which may have an effect on the system integration and compatibility.Notably, despite the demonstrated ultrahigh sensitivity and versatile multiplexing applications, the robustness of bio-MEMS systems must be enhanced prior to their use for on-site airborne coronavirus detection.5.3301 The highly sensitive and selective detection of a trace amount of airborne pathogens in this diverse aerosol background remains challenging. In addition to improving the sensitivity, on-site bioaerosols detection demands effective strategies to minimize non-specific interference. In this section, we consider the main aerosol interference factors, including physical disruption from airborne particles, chemical inhibition of redox-active species, and biological interferences.Aerosols represent complex chemical components in the air, mainly including metal ions, inorganic particles, organic carbon, and bioaerosols.17,302 Hong and co-workers developed a microfluidic inertial separator to simultaneously separate airborne pathogens by size.205 Airborne viruses, with dimensions less than 500 nm, can be purified and subsequently measured using a real-time analyzer. For size-based purification, active separation techniques employing acoustophoretic and dielectrophoretic techniques in a microfluidic system can also be utilized.303Sample pretreatment and direct size-dependent separations may be the simplest method for eliminating the impact caused by insoluble dispersed particles. Jang and co-workers proposed an on-site airborne pathogen biosensing system that integrated multilayer filtration pads with different pore sizes to purify the collected airborne pathogens and eliminate the interference from dust particles.173,174,304 To this end, scavengers for redox-active species and radicals may be potentially used to reduce the oxidation of biomolecules and biosensing recognition sites.181 As shown in 2−, H2O2, 1O2, ONOO−, and ONOOH and found that NaN3 (scavenger for 1O2), uric acid (for ONOO−), and ascorbic acid (for ONOOH) could be good candidates to preserve the viability of collected airborne pathogens such as viruses.173 Using appropriate chemical scavengers in the aerosol-to-hydrosol sampling and bioaerosol elution process may preserve the pathogens as well as their protein structures and nucleic acid sequences, thereby enhancing the biosensing accuracy and preventing the underestimation of airborne concentrations.Redox-active species, such as ROS, RNS, and transition metal ions, may inhibit the effective recognition of airborne pathogens by damaging the activity and functionality of recognition molecules.229,272–274 Intelligent biosensor design could be another significant strategy to tackle non-specific molecular interference. For instance, signal or molecular switch system and self-referenced transduction can effectively improve the bioaerosol sensing accuracy in a complex liquid matrix.228 The signal/biomolecular switch biosensing system is composed of functional proteins or nucleic acids that undergo conformational or functional changes upon interaction with the target pathogens. The distinct bioactivities of the biochemical switches, such as the cleavage activity of an enzyme or the photon emission of a bioluminescent protein, can be engaged when the biosensing targets contact the designated functional units.305,306 Based on the switches, non-specific molecules, even if bonded onto the biosensor surface, cannot activate the specific biofunctions, thereby avoiding the interference of complex non-specific background aerosols. As a type of robust functional biomolecules, CRISPR/Cas proteins can be used as enzymatic switches for detecting different pathogens. Additionally, site-specific restriction enzymes, such as nicking endonucleases, could also be employed to design biomolecular switch biosensors. For instance, endonuclease IV, which cleaves the apurinic/apyrimidinic (AP) sites in double-stranded nucleic acids, can be used as a biomolecular switch for airborne virus detection.18,20 This cleavage function is only activated when the pathogenic sequences are present and bind to the oligonucleotide probes, thereby avoiding false signals caused by non-specific binding events. Another prominent example of novel biomolecular switches is the de novo designed bioactive protein biosensors, as shown in 252,253Antifouling coatings represent one of the most useful chemical approaches. Readers could refer to these recently published articles, which have systematically summarized the antifouling technologies for biosensing applications.While there are a growing number of promising developments of effective strategies for tackling non-specific aerosol interference with sample pretreatment, chemical scavengers, and intelligent biosensor designs, on-site biosensing implementation and robustness validation are still scarce. To further optimize the antifouling strategies and enhance the bioaerosol sensing robustness, more real-world implementation works including bioaerosol sample collection and on-site quantification should be conducted.5.4.14,17,19,307–310 Choi and co-workers developed a fully integrated optofluidic SERS biosensing platform for the simultaneous detection of airborne pathogens, as shown in 286 The microfluidic-based liquid impingement sampler with the two-phase flow  was employed to collect the airborne bacteria. Colloidal silver nanoparticles (AgNPs) in the collection liquid could be directly adsorbed onto the target biomolecules in the microfluidic mixer and then enhanced the Raman spectroscopic signal in a continuous-flow manner. The sensitivity of this real-time and continuous bioaerosol sensing system was reported to be relatively low at 100 CFU per mL.From bioaerosols collection to the interpretation of the sensing results, airborne pathogens detection is generally carried out following specific steps in practical on-site and rapid infection risk assessment. This process mainly includes four parts: (1) aerosol sampling and enrichment, (2) pretreatment and delivery, (3) quantitative biochemical analysis, and (4) interpretation of bioaerosol sensing results. Some pioneering and recent research works reported the integration of different functional units for realizing on-site airborne pathogen detection in a continuous and real-time manner /m3.The combination of a filtration-based airborne pathogen sampling system and a lateral flow sample delivery system represented another instance of compatible and field-deployable airborne pathogen sensing system.g device , propose310 This electrochemical biosensing system utilized the magnetic nanoparticles and functionalized antibodies to capture the collected airborne viruses. By leveraging the external magnetic fields, these magnetic nanoparticles not only facilitated the virus enrichment on the electrochemical sensing surface but also erased all the biomolecules and pathogens after detection, thus achieving the reuse of the electrochemical sensor.Another on-site airborne pathogen biosensing example, as shown in 20 In addition to direct measurement of the airborne pathogen exposures with high spatiotemporal resolution, the biosensing signal can be translated to probabilities of infection risk and estimate maximum exposure durations to an acceptable risk threshold in different environmental settings.More recently, a condensation (hygroscopic growth)-assisted bioaerosol collection and plasmonic photothermal sensing (CAPS) system was developed for the on-site quantitative risk analysis of pathogen-laden aerosols.310,311 Therefore, in the follow-up work, the automation of the systems also needs to be further improvement. Regarding the capability of telecommunication and connectivity, Xue et al. proposed an integrated Bluetooth unit to directly upload the viral sensing results to the end-users , the connectable on-site airborne virus sensing systems are poised to enable rapid infection risk assessment and evidence-based public health intervention. Another potential issue is that the majority of existing airborne pathogen biosensing systems cannot differentiate their viability and infectivity. Currently, there are several studies that utilized off-site and culture-based methods to investigate the viability of airborne viruses from different environments scenarios including patient wards and private car.51,211 There is still a high demand for the development of on-site biosensing technologies that can directly quantify viable airborne pathogens.Notably, it is found that the sensitivity of current on-site airborne pathogen biosensing platforms still needs to be further improved. Although detection sensitivity can be further improved through bioaerosol enrichment by extending the aerosol sampling duration, the potential physiochemical impact during the long sampling-to-biosensing process may also lead to a non-negligible loss of pathogen integrity and functionality. In addition, long sampling duration would deteriorate the time resolution of the biosensing system and jeopardize the capability for time-sensitive risk assessment. Some of the proposed pathogen aerosol sensing systems still require manual operations such as bioaerosol elution, pathogen lysis, and sample delivery.nd-users .247 By f618,41,310 Therefore, there is a high demand for incorporating sensitivity improvement strategies into airborne pathogen sensing systems. One of the most effective ways is to amplify the biosensing response triggered by the presence of pathogen targets.312,313 In addition, the selective amplification of biosensing targets or signals could also diminish the interference caused by complex and diverse background components.20Improving the sensitivity of detecting airborne pathogens can significantly enhance the reliability of transmission risk assessment and evidence-based public interventions.312,314–316 This section focuses primarily on two essential physiochemical approaches, namely, (1) enzyme catalysis-based amplification approaches and (2) nanomaterial-based amplification approaches. These amplification techniques could be further developed into “plug-and-play” modules for numerous on-site airborne pathogens and bioaerosols analyses with enhanced sensitivity and robustness.Numerous amplification strategies have been proposed and implemented to enhance the sensitivity.6.1317 While in a PCR system, the short complementary oligonucleotides primers can be elongated by the polymerase enzymes when they specifically bind to the target sequences. By repeating the hybridization–polymerization–dehybridization process, the biosensing targets can be rapidly replicated and thereby realize highly sensitive pathogen detection.239 Inspired by these two conventional amplification concepts, enzymatic and catalysis-based amplification strategies can be adapted for on-site airborne pathogen detection.310,318–321Enzymatic and catalysis-based amplification strategies represent a fundamental concept for sensitivity enhancement, which leverages the chemical reaction at the biosensing step to amplify the transducing signal or the detection target. Enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) are two examples that amplify the sensing signal and detection target, respectively, using enzymatic chemical reactions. Antibody-conjugated enzymes can be specifically immobilized onto the ELISA wells by recognizing epitopes and triggering a chemical reaction that converts the color of appropriate probing substances, such as 3,3′,5,5′-tetramethylbenzidine (TMB) or 2,2′-azino-bis(3-ethylbenzothiazole-6-sulfonic acid) (ABTS).6.1.1315,322 PCR, as the ‘gold standard’ of detecting airborne pathogens, has demonstrated superior sensing reliability.239 However, the thermal cycling process with bench-top PCR devices limits its applicability in the field of rapid on-site coronavirus quantification. In contrast, isothermal amplification methods such as recombinase polymerase amplification (RPA), rolling circle amplification (RCA), and loop-mediated isothermal amplification (LAMP) can be alternative candidates for rapid and on-site airborne pathogen sensing applications.315,323–327 Ganguli and co-workers demonstrated an isothermal LAMP-based biosensing system for highly sensitive pathogens detection.328 Using a microfluidic-based isothermal amplification cartridge and a smartphone-based fluorescence readout system, this amplification-based system achieved a superior detection limit of 50 viral RNA copies per μL within 30 min. Jiang and co-workers integrated the LAMP isothermal amplification system into a hand-held device for rapid and on-site airborne virus detection.41 The pathogen-laden aerosols, collected by an liquid impactor, can be directly pre-treated  and isothermally amplified on a paper-based analytical system. The whole on-site sampling-to-biosensing process took only about 1 h and provided a detection limit of 1 TCID50 for the H1N1 influenza virus model.As highly specific biomarkers, nucleic acids of airborne pathogens are ideal replication targets for enzymatic amplification and achieving enhanced sensitivity and selectivity.et al. harnessed the voltametric electrochemical biosensor and isothermal RPA approach to rapidly detect the amplified viral targets with a detection limit of 1000 viral copies per μL.319 Moreover, taking advantages of the miniaturized microelectronic components, the on-chip RPA system can be operated using the human body temperature heating, as shown in By further incorporating isothermal amplification strategies and novel transducing techniques, the integrated airborne pathogen sensing system could further improve the biosensing performance and the integration level of the whole system. Kim 329,330 Instead of using conventional heating components such as Peltier-block heater, photonic PCR devices employed plasmonic photothermal effect to achieve ultrafast thermocycling and efficient nucleic acid amplification.329 As shown in i.e., SARS-CoV-2) using plasmonic nanoparticles as an ultrafast heating source.331 This prototype could simultaneously measure three samples within 17 min and achieve a detection limit of 3.2 viral copies per μL. In 2021, Kang and coworkers further integrated the nanophotonic PCR system onto a microfluidic on-chip PCR platform.332 This miniaturized plasmofluidic PCR system was able to complete 40 thermocycles of amplification within 5 min. Therefore, by further integrating the microfluidic-based aerosol sampling and pretreatment system, this ultrafast and sensitive amplification strategies can be potentially used for on-site airborne pathogen detection and infection risk assessment.In addition, the development of novel nanophotonic technologies also offers a convenient avenue for amplification-based bioaerosols detection. Plasmonic-enhanced photothermal effect can be utilized as a precise and efficient heating source to promote the enzymatic amplification in polymerase chain reaction.333 The airborne redox-active species , metal ions , and biological compounds  might inhibit the functionality of the enzyme and interfere with the quantification of the airborne viruses.333,334 Therefore, it is also important to incorporate effective strategies to further purify the pathogenic targets and remove the amplification inhibitors.335–338 For instance, the microfluidic-based bioaerosol purification strategies (Section 4.5) and chemical scavengers for removing airborne redox-active compounds (Section 5.3) can be utilized to ensure a stable and effective enzymatic amplification.However, the integration of enzymatic amplification strategies for airborne pathogen detection also faces challenges such as the interference from complicated aerosol background.6.1.2310,339,340 In addition, the highly specific nucleic acid probes could also be used to immobilize the oligonucleotide-linked enzymes for conducting ELISA-like signal amplification, as shown in 341Beyond target-amplification options, a further interesting approach to increase the sensitivity of airborne pathogen detection is to amplify the biosensing readout signal through enzymatic and catalytic activities. Instead of using conventional benchtop readout systems, the signal amplification concept in ELISA can be further integrated with microfluidics and novel biosensors to further improve the sensing swiftness, sensitivity, and robustness.342 Hybridization between the pathogen sequence to Cas-crRNA strand ensures a high recognition specificity and subsequently activates the cleavage function of the Cas protein to amplify the biosensing signal by indiscriminately cleaving surrounding sequences .343 For instance, Fozouni and co-workers proposed a portable and smartphone-based CRISPR/Cas13 biosensing system for highly sensitive viral RNA detection  in various complex real-world samples.CRISPR/Cas-based technologies represent another type of promising strategies for biosensing signal amplification.etection .225 Thisvia cyclic cleavage in order to accomplish “switch-on” or “switch-off”-based signal amplification.18,345 For instance, in a “switch-on”-based amplification system, the fluorophore-quencher pairs can be continuously processed by restriction endonuclease after hybridization with the virus sequences.18 The localized nanophotonic heating could facilitate the entire isothermal process for highly efficient signal amplification, including the dehybridization of cleaved short oligonucleotide and promotion of enzymatic reactivity. Compared to the non-amplification results with the same biosensing platform, this signal amplification strategies significantly improved the sensitivity by up to two orders of magnitude for airborne pathogen detection.Site-specific nucleic acid cleavage enzymes such as nicking enzymes and restriction endonucleases have also been utilized for biosensing signal amplification. These site-specific enzymes could selectively cleave designed probes in situ on the transducing surface is considered as an alternative approach to magnify the readout signal and improve the bioanalytical sensitivity.346,347 Generally, the initiating molecules are conjugated to protein or nucleic acids probes, which can specifically recognize the pathogen targets and allow localized chemical reactions to subsequently proceed. Polymerization-based signal amplification can be achieved by harnessing a variety of different chemical reactions, including atom transfer radical polymerization (ATRP), fragmentation chain transfer polymerization (RAFT), ring-open metathesis polymerization (ROMP), and enzyme-mediated redox polymerization.348,349 Recently, Kim and co-workers reported a novel exponential signal amplification strategy based on the photo-initiated redox autocatalysis, wherein the photocatalyst (Eosin Y) amplified the biosensing signal by activating a nonfluorescent Eosin Y derivative (EYH3−) under green light illumination.347 Additionally, Eosin Y amplification was coupled with another photo-sensitive reaction, i.e., the oxidative polymerization of 3,3′-diaminobenzidine (DAB), so as to yield different forms of signals and benefit the sensitive detection of antigenic epitopes of pathogens. Gurnani and colleagues have described an oxygen-tolerant RAFT polymerization platform based on a modified Fenton reaction that is initiated by the iron-reducing bacterial species Cupriavidus metallidurans.350 Although this RAFT method was proposed for producing well-defined polymeric materials, this concept also demonstrated high potential for highly sensitive viable pathogens detection.351Target-triggered and polymerization-based signal amplification performed 6.2Nanomaterials are attractive candidates for signal amplification and sensitivity enhancement in novel pathogen biosensors due to their unique optical, electrical, and magnetic properties as well as enhanced reactivities. On the basis of amplification principles, nanomaterials for bioaerosol sensing amplification have been divided into four distinct classes: nano-catalysts .360,361 For instance, based on the Förster resonance energy transfer (FRET) effect, the designed conjugation of functionalized QDs and AuNPs can be used to monitor the viral spike-ACE2 bindings.362 Apart from the direct observation of the fluorescence reaction generated by FRET, the amplified energy transfer signal can also be quantified by highly sensitive approaches such as LSPR and electrochemical sensors  and upconverting nanoparticles (UCNPs) can be used as effective nano-reporters and nano-magnifiers to enhance the transducing signal or individually provide a secondary readout. sensors .18,361,3364,365 For example, colloidal silver nanoparticles can be utilized to capture airborne pathogens in a microfluidic system and subsequently employed as nano-magnifiers to amplify the Raman signal for the real-time quantification of bacteria such as S. epidermidis, M. luteus, E. hirae, B. subtilis, and E. coli.286 Meanwhile, the photothermal effect with plasmonic nanomaterials can also improve the biosensing performance by tackling the diffusion limit in localized biosensing surface.283,366,367 As shown in Nanomaterials, which may possess unique optical or electronic properties, can be directly used as nano-magnifiers to amplify the biosensing signal. In particular, by enhancing light-matter interactions, nanoparticles can achieve enhanced Raman scattering (SERS) and fluorescence emission (SEF) at the biosensing surface .364,365 314,323,328,347,352,354,368–370 These solutions have the potential to be further developed as “plug-and-play” components for adaptable biosensing systems, thereby achieving higher sensitivity, selectivity, accuracy, and swiftness for bioaerosol sensing applications. Meanwhile, these integrated biosensing system could be employed as direct exposure measurement devices for the highly reliable risk assessment of airborne pathogen transmission. Compared with indirect modelling approaches, novel biosensors will play a crucial and complementary role in the research of bioaerosol transmission, public health intervention, and disease prevention.The in-depth investigation and discussions of how to use biological enzymes or nanoparticles to improve the biosensing performance have been presented in numerous research works recently.7e.g., by causing respiratory infection or sick building syndrome. This does require more systematic research works by researchers from different communities and aspects.Bioaerosol researches have been conducted for decades. However, COVID-19 outbreak has once again brought to light the fact that bioaerosols and airborne pathogens play a critical role in the transmission of specific diseases. A majority of environmentalists, clinicians, and epidemiologists underestimated the potential of bioaerosol transmission in the early stages of the COVID-19 outbreak. This has forced us to reflect again on whether common bioaerosols also significantly impact our well-being and health condition more universally in daily lives, SARSVAC” criteria for evaluating the biosensing system, which means that the biosensing system should not only be Sensitive, Accurate, Robust, and Swift but also have the potential to be Versatile, Automated, and Connectable for implementation in distributed biosensor networks for the large-scale risk assessment of bioaerosols. Notably, the present review attempts to sketch the early stage of what is expected to become a long journey, as shown in On-site airborne pathogen detection with novel bioaerosol sampling and quantitative analysis systems could provide a straightforward approach to achieve swift airborne pathogen exposure and transmission risk assessment. In this article, we summarized the crucial physicochemical properties that should be considered in on-site airborne pathogen detection. We also provided a thorough analysis of recent developments in the on-site pathogen-laden aerosol sampling and biosensing strategies with the consideration of these features. For instance, a miniaturized aerosol-to-hydrosol sampling device with a high ESEC performance can be employed for fast bioaerosol sampling. Subsequently, the downstream biosensing system could provide sensitive and reliable quantification results for estimating the airborne pathogen exposure and probability of infection risk. More importantly, by considering the requirements for on-site airborne pathogen detection and risk assessment, we also establish the “7.1SARSVAC” criteria is required.To genuinely achieve the on-site quantification of trace quantities of contagious airborne pathogens, additional development under the “e.g., microfluidic bioaerosol samplers. This indicates aerosol sampling techniques that are incapable of handling pathogen-containing air samples of several liters per min have a significantly reduced utility in many practical scenarios.(1) Choosing an appropriate bioaerosol sampling strategy for subsequent detection. The possible loss and inactivation of the airborne pathogens may lead to severe underestimation in quantitative chemical analysis. Particularly, some bioaerosol sampling techniques, such as electrostatic and filtration-based samplers, may damage the functionality of the pathogen recognition sites, such as the nucleic acids or proteins . These d(2) Thoroughly considering the environmental impacts on the pathogen-laden aerosols during the transmission, sampling, and biosensing processes. The ideal “sampling-to-biosensing” procedure can be quick and conducted on-site. However, the airborne pathogens may still be significantly impacted by external environmental conditions such as RH, temperature, irradiation, aerosol pH, and airflow, leading to an underestimation of the transmission hazards. Accordingly, real-time environmental sensors can be deployed for monitoring ambient conditions such as temperature, irradiations, and RH (see Section 2.4) throughout the on-site bioaerosol sampling and transducing procedures. This allows for an estimation of pathogen degradation and inactivation.(3) Improving the physical collection efficiency for nanoscale bioaerosols and considering the potential “penetration/escape window” in the pathogen-laden aerosol sampling. Pathogen-laden aerosols may be distributed in a wide aerodynamic size range from 20 nm to 0.2 mm . Therefo(4) Considering the viability and infectiousness of airborne pathogens and developing on-site biosensing technology for effective bioaerosol risk management. Current biosensing techniques that rely on recognizing nucleic acids and protein molecules primarily quantify all airborne pathogens, including inactivated pathogens and their fragments. However, these detection results do not inherently correlate with the true risk of infection transmission because only viable pathogens can induce infection. Few biosensing technologies currently exist that can swiftly distinguish viable pathogens and characterize their infectiousness. Using cutting-edge biosensing technologies, quantitative viability and infectiousness results should be determined on-site as a crucial foundation for precise risk assessment.via direct biosensing measurements.(5) Investigating the concentration of pathogens in airborne particles as a function of aerosol size and their ability to initiate infection . This si(6) Considering the ESEC of a chosen bioaerosol sampler. A high aerosol enrichment coefficient (ESEC) can enhance the biosensing swiftness and sensitivity for on-site airborne pathogens detection. Generally, ESEC can be improved by integrating high flowrate sampling units and miniaturized microfluidics. Additionally, a multifunctional microfluidic system can also be utilized as an effective method for rapidly pretreating and transporting the collected bioaerosols for quantitative chemical analysis.(7) Improving the sensitivity and reliability of the biosensors by leveraging the amplification strategies. Highly sensitive airborne pathogen detection can be achieved by improving the intrinsic biosensor designs  Long-term utility and regeneration of biosensor systems for multiple measurements. Although many different biosensors have demonstrated excellent sensing performance for airborne pathogen quantification and risk assessment, many are still stuck with disposable cartridges for a single or few times of use. In consideration of the practical monitoring applications and sustainability of biosensors over extended periods of time, this could become a key barrier to widespread adoption. One way to improve the long-term utility that is suitable for certain airborne pathogen biosensors is to enable sensor-regeneration. Multiple approaches are potentially available for biosensor regeneration. In the literature, pH control is the most commonly used approach for the regeneration of antigenic epitopes-based biosensors. Altering the temperature and ionic strength as well as using strong detergents have been demonstrated to regenerate nucleic acid-based biosensors. However, one current challenge pertains to the absence of a clearly defined criteria for successful regeneration. Hence, it is difficult to assess the effectiveness of different regeneration procedures for detecting airborne pathogens. In addition, affinity-based bioreceptors possessing excellent binding affinity offer low detection limits for sensitive airborne pathogen quantification, which implies that the regeneration of the biosensors will be extremely difficult. Therefore, how to achieve high binding affinity to the pathogenetic analyte in a reversible fashion represents another critical challenge in the field.(9) Developing a cost-effective and user-friendly on-site sensing system for connectable sensor networks. With the aid of wireless communication and IOT technologies, low-cost and user-friendly biosensors could be deployed in numerous locations, covering potential hotspots and forming high-density networks to provide the real-time mapping of the airborne pathogen distribution. The extreme version would consist of personal bioaerosol sensors that individuals can carry and use to monitor the bioaerosol exposure in their immediate vicinity, allowing for personal risk mitigation. IoT bioaerosol sensors have the potential to revolutionize the monitoring and management of bioaerosol exposures. As technology continues to evolve, these sensors should be made more intelligent and affordable, making them available to a wider range of individuals.e.g., 0.1%, 1%, 5%, and 63%) can be estimated. The World Health Organization (WHO) suggested that face-to-face contact with a case within 1 m and for >15 min can be identified as a close contact and having a potential risk of infection.371 Based on the metanalysis results, the infection risk (probability) for this reference scenario can be estimated to be 1%.136 In addition, the airborne virus concentration based on the indirect modeling approaches ((10) Translating the biosensing results to potential infection risks and health impact. The concentration of airborne pathogens or bioaerosols detected by an on-site biosensing system can be used to estimate the probability of individual risk. proaches  were als(11) Estimating the uncertainties and errors in on-site airborne pathogen detection. The majority of current biosensing methods for measuring ambient pathogen concentration were based on recognizing nucleic acid sequences or antigenic proteins, without considering pathogenic infectivity and viability. Risk assessment based on these results may lead to the overestimation of airborne pathogens concentration and infection risk level. Therefore, future research should incorporate biosensors that can differentiate the infectivity and viability of airborne pathogens in order to accurately assess the amount of viable pathogens as opposed to the total number of nucleic acid copies or protein concentrations. These uncertainties caused by the diversity of pathogen infectivity and viability should be fully considered in subsequent risk assessment.7.2On-site exposure measurement and evidence-based infection risk assessment are growing to be an important field for investigating the health impact of airborne pathogens. An accurate on-site bioaerosols detection system not only bridges the gap in the conventional understanding of airborne pathogen spreading but also provides a promising avenue for swift public health intervention and decision-making. Despite their availability, on-site airborne pathogen detection and quantification methods have not been widely deployed in field applications due to technical or expense considerations. Herein, we further discuss the future development opportunities and challenges for evidence-based risk assessment.e.g., pathogen inactivation rate, aerosol deposition rate, and quanta conversion factors), thereby improving the accuracy of the model-based exposure assessment.20(1) Optimizing the emission and exposure model through direct on-site measurement. Currently, epidemiological information from previous infection cases is generally utilized for estimating model-based bioaerosol emissions and individual exposure. The individual differences, such as pathogen shedding and the bioaerosols emission, are generally overlooked in the model-based methods and therefore cause high uncertainties. This explains why the simulated pathogen exposure and the actual measured values can vary by one to two orders of magnitude. The on-site airborne pathogen detection can be used as a direct verification technique to further optimize the models and the calculation parameters  Optimizing the dose–response model through direct on-site airborne pathogen measurement. On-site airborne pathogen detection with biosensors can provide accurate exposure information with high spatiotemporal resolution for a specific infection case. By calculating and fitting the infection correlation, a pathogen-specific dose–response model can be effectively retrieved. With further validation, this on-site airborne pathogen measurement approach may become a viable alternative to the human-challenge (deliberate human infection) approach. In addition, establishing a reliable and accessible database for dose–response models of numerous airborne infectious diseases is also essential. The IDs review , but fur(3) Employing a biosensing system with a high spatiotemporal resolution to investigate the aerodynamic nature of airborne pathogens. Currently, the majority of cases of airborne pathogen transmission have been discovered through an indirect method by ruling out other fomite- or contact-based infections. The absence of direct on-site biosensing instruments and straightforward aerodynamic observations for airborne pathogens is one of the most importance factors. By deploying on-site biosensors for swift and real-time bioaerosol detection, the aerodynamic properties of pathogen-laden aerosols can be more accurately characterized. To accomplish this goal, however, further development in improving the sensing performance and integration of bioaerosol sensing systems is still highly demanded.3 h−1).20 In future work, the integrated on-site bioaerosol sensing system and networks can be used to estimate personal infection probabilities, occupancy limit, and maximum exposure period in an indoor environment.(4) Directly calculating the infection risk or bioaerosols impact using the biosensing results and advising the maximum exposure times for particular situations with a potential airborne pathogen presence. Though many integrated “sampling-to-biosensing” systems have succeeded in on-site bioaerosols and airborne pathogens quantification, the biosensing results have not been translated into an interpretable risk level and utilized for epidemic management. For instance, the high spatiotemporal airborne pathogen concentration measured by biosensors can be potentially used to estimate maximum tolerable exposure durations in different risk scenarios. Based on the bioaerosol sensing results, a recent work reported that the maximum exposure time in a high-risk COVID-19 patient ward was estimated to be about 46 min for a susceptible individual wearing an FFP2 facemask and engaging in low-intensity activity  Combining the direct on-site airborne pathogen measurement method and the indirect exposure modelling approach for smart decision making. A highly accurate on-site biosensing system could provide a straightforward pathogen exposure information. However, it may still be unaffordable or impractical for large-scale biosensing network. Indirect modelling approach could be more cost-effective and affordable. However, inter-individual differences on pathogen emission are generally not considered. Therefore, to carry out an effective risk assessment for transmission mitigation, it is essential to investigate how to incorporate both direct measurement and indirect modelling methodologies in an integrated smart sensing system. The optimization of risk assessment should consider more significant factors, such as vaccination and health status.(6) Predicting infection, disease severity, and morbidity using a distributed on-site airborne pathogen sensing network. As a crucial spreading route for many infectious diseases, the concentration or emergence of airborne pathogen can be used as an effective factor to predict infection, disease severity, morbidity, and hospitalization rates. Direct measurement findings and a bigdata-based forecasting system could assist in medical facilities match potential patients with the proper degree of care capacity in order to more effectively manage their limited medical resources amid a spike in infection cases. As a result, the research priority may be used to further interpret the results of on-site and real-time airborne pathogen sensing from various locations, which may call for the combined efforts of epidemiologists, analytical chemists, data scientists, health care providers, and experts on air quality.20 If AI-based decision-making systems can be incorporated into networked bioaerosol sensors for intelligent and automated risk assessment, a more credible and trustworthy early-warning system will be realized.(7) Developing a decision support system by integrating artificial intelligence (AI) and an on-site pathogen sensing system . AI deciIn conclusion, we strive with this review not only to provide comprehensive knowledge and insights about the recent advancements in the on-site airborne pathogen biosensing but also to further stimulate more in-depth reflection and research works in related fields. In recent years, significant progress has been made in understanding airborne transmission routes and developing biosensing technology, particularly in the areas of point-of-need detection and environmental monitoring. However, there is still considerable room for improvement in integrating rapid bioaerosol samplers and conducting on-site risk assessments for different types of airborne pathogens or bioaerosols. As mentioned, this review article only provides a snapshot for an early stage of what is expected to become a long scientific journey in on-site bioaerosol assessment. We look forward to the advancements in on-site airborne pathogen sensing and their applications in early-stage risk assessment. We intend for this review article to provide a roadmap for identifying the main research priorities and purposefully advancing on-site biosensing technologies. More significantly, the reviewed pathogen-laden aerosol sampling and biosensing technologies could also provide a promising avenue for conducting critical bioaerosol risk assessment and protecting the public health especially in on-site bioaerosols measurement scenarios such as the monitoring of high-risk pathogens at the check-in point of airplanes or cruise ships, defending weaponized pathogen aerosols, screening influenza variants at hospitals or other healthcare settings, and monitoring sick building syndromes at different workplaces.There are no conflicts of interest to declare.CS-052-D3CS00417A-s001