Device for collecting exhaled air condensate and respiratory virus home detection method

Abstract

The invention discloses a device for collecting exhaled air condensate and a respiratory virus home detection method, the device comprises a main body, the main body comprises a semiconductor refrigeration module, an airflow channel at least partially arranged in the semiconductor refrigeration module, and a disposable unit detachably connected to an inlet of the airflow channel; a label-free electrochemical sensor for receiving the exhaled air condensate sample from the body; and the analysis module is an application program loaded on electronic equipment, a machine learning algorithm is built in the analysis module, and the analysis module is in communication connection with the unmarked electrochemical sensor and is configured to receive the electric signals of the unmarked electrochemical sensor and perform automatic analysis, comparison and result output. According to the invention, rapid, stable and non-invasive sample collection without pre-cooling can be realized, and rapid detection of respiratory viruses can be completed in a home environment.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection and sensing technology, specifically to a device for collecting exhaled breath condensate and a method for home detection of respiratory viruses. Background Technology

[0002] Exhaled breath condensate (EBC) is a liquid sample collected by cooling exhaled air and then condensing the aerosols from the lower respiratory tract. EBC is rich in biochemical components from the lungs and airways, including proteins, inflammatory factors, electrolytes, and biomarkers such as pathogens (e.g., viral particles). Because EBC is derived directly from alveolar lining fluid, its compositional changes can more accurately reflect the pathophysiological state of the lungs and airways, making it superior to nasopharyngeal swabs or saliva samples in reflecting lung infection activity. Furthermore, EBC collection is non-invasive, repeatable, and highly tolerable for patients, avoiding the discomfort, operational errors, and inconsistent sample quality and false negative risks associated with invasive sampling (such as nasopharyngeal swabs). Therefore, it is considered a highly promising medium for screening and diagnosing respiratory diseases.

[0003] Despite the advantages mentioned above, the widespread application of EBC in clinical settings, especially in home settings, still faces a series of key technical challenges: 1. Insufficient Standardization and Portability of Sample Collection: Early development of EBC collection technology relied on traditional methods such as cold trap condensation. Existing commercial equipment (such as EcoScreen and Rtube) is generally bulky (weighing over 20 kg) and lacks portability. These devices typically require extremely low cooling temperatures (e.g., below -10°C) or pre-freezing to operate effectively, resulting in low energy efficiency and poor thermal stability. Standard collection procedures are time-consuming (usually exceeding 10 minutes), easily causing dry mouth and saliva contamination. Furthermore, collection efficiency and stability are significantly affected by ambient temperature, humidity, and the user's breathing pattern, leading to poor reproducibility and difficulty in achieving standardized collection. Although some mask-based condenser designs have recently emerged, they still require pre-cooling in a refrigerator and have long collection times, failing to fundamentally solve the problems of convenience and immediacy.

[0004] 2. Subsequent testing methods cannot meet the needs of rapid home diagnosis: Currently, the analysis of EBC samples mainly relies on laboratory techniques, such as enzyme-linked immunosorbent assay (ELISA) or quantitative reverse transcription polymerase chain reaction (qRT-PCR). While these methods offer good specificity, they have inherent limitations, including complex operation, lengthy processing times, and the need for expensive specialized equipment and skilled technicians. They cannot meet the urgent need for rapid on-site testing, especially in home testing scenarios where speed and convenience are crucial. Even as the gold standard, the accuracy of qRT-PCR is susceptible to the quality of EBC sample collection.

[0005] 3. Lack of integrated detection systems for home environments: Existing technologies typically separate sample collection from subsequent testing and analysis, lacking a miniaturized, integrated system that combines efficient, non-invasive collection, highly sensitive detection, and intelligent result interpretation. The market urgently needs a home-use device capable of achieving a closed-loop "sampling-detection-analysis" process to improve the universality, ease of use, and reliability of testing.

[0006] 4. The sensor's performance is difficult to match the specific characteristics of the EBC sample: Electrochemical sensors are considered ideal platforms for point-of-care detection due to their high sensitivity, fast response, and ease of miniaturization. However, existing sensors, especially electrochemical immunosensors for complex biological sample detection, mostly rely on exogenous redox media to generate signals. Their signal response may be negatively correlated with the target analyte concentration and is easily affected by experimental conditions. More importantly, in real-world samples such as EBCs (electrochemical biomarkers) with extremely low biomarker concentrations and abundant matrix interference, the selectivity, anti-interference capabilities, and stability of existing sensors remain insufficient, and their recognition performance and reliability need improvement.

[0007] In summary, existing EBC collection technologies suffer from drawbacks such as bulky equipment, low efficiency, and difficulty in standardization; while subsequent detection methods are reliant on laboratories, complex in operation, time-consuming, and unable to handle low-concentration samples. Both factors contribute to the current lack of a complete solution suitable for home environments, enabling early, rapid, non-invasive, and highly sensitive detection of respiratory viruses. Therefore, developing a portable, integrated platform that combines rapid and stable collection technology with a highly sensitive label-free detection method is of great significance for home screening and prevention of respiratory infectious diseases. Summary of the Invention

[0008] In order to overcome the shortcomings of the prior art, one of the objectives of the present invention is to provide a device for collecting exhaled condensate, which can achieve rapid, stable and non-invasive sample collection without pre-cooling, and complete rapid detection of respiratory viruses in a home environment.

[0009] The second objective of this invention is to provide a home-based method for detecting respiratory viruses, which enables rapid, stable, and non-invasive sample collection without pre-cooling and allows for rapid detection of respiratory viruses in a home environment.

[0010] To achieve one of the objectives of this invention, the following solution is adopted: A device for collecting exhaled breath condensate, comprising: The main body includes a semiconductor cooling module, an airflow channel at least partially disposed inside the semiconductor cooling module, and a disposable unit detachably connected to the inlet of the airflow channel; the airflow channel is formed by a thin tube coiled inside the semiconductor cooling module, used to condense exhaled gas to obtain an exhaled gas condensate sample; the disposable unit includes a mouthpiece and a connecting tube, the mouthpiece being connected to the connecting tube, and the connecting tube being connected to the inlet of the airflow channel; A label-free electrochemical sensor for receiving exhaled condensate samples from the subject is constructed by depositing a carboxylated single-walled carbon nanotube film in the working area of ​​a screen-printed electrode and immobilizing angiotensin-converting enzyme 2 receptor on the film using a 1-pyrene butyrate N-hydroxysuccinimide crosslinking agent. The analysis module is an application program installed on an electronic device. The analysis module has a built-in machine learning algorithm and is communicatively connected to the label-free electrochemical sensor. It is configured to receive the electrical signals from the label-free electrochemical sensor and perform automated analysis, comparison, and result output.

[0011] Furthermore, the length of the main body is no greater than 25 cm, the width is no greater than 20 cm, the height is no greater than 20 cm, and the weight is no greater than 2 kg.

[0012] Furthermore, the design of the airflow channel ensures that the temperature fluctuation of its condensation area is less than 2°C under the exhaled airflow conditions of a febrile patient.

[0013] Furthermore, the main body is configured to collect at least 50 µL of exhaled condensate within 30 seconds of continuous, gentle exhalation by the user; and to collect at least 500 µL of exhaled condensate within 5 minutes of exhalation.

[0014] Furthermore, the label-free electrochemical sensor has a detection limit of no more than 10 fg / mL for SARS-CoV-2 pseudovirus particles.

[0015] Furthermore, the analysis module is configured to use the rate of change of current response value (I-I0) / I0 as the analysis parameter, where I0 is the initial current value before the sample is added and I is the response current value at a specific time after the sample is added; and to compare this parameter with a threshold predetermined by clinical samples.

[0016] To achieve the second objective of this invention, the following solution is adopted: A method for home testing of respiratory viruses using a device for collecting exhaled breath condensate as described in one of the objectives of this invention includes the following steps: Sample collection steps: The user exhales into the device for collecting exhaled condensate through the disposable unit for 30 seconds to 5 minutes to obtain an exhaled condensate sample; Direct detection step: Take no more than 5 µL of the exhaled breath condensate sample and drop it directly onto the electrode surface of the label-free electrochemical sensor. Detect the current-time curve and obtain the signal value (I-I0) / I0 within 60 seconds using chronoamperometry in phosphate buffer. Intelligent interpretation steps: The signal value is input into the analysis module, which is automatically analyzed by the machine learning algorithm built into the analysis module, and outputs a positive, negative or retesting result within 60 seconds.

[0017] Furthermore, during the sample collection step, the user exhales gently at a normal breathing rate.

[0018] Furthermore, the entire process of this home-based respiratory virus testing method, from sample collection to result output, takes less than 8 minutes.

[0019] Furthermore, this home-based respiratory virus testing method can effectively detect samples during the infection window period, asymptomatic infection period, and antigen-negative samples, with a clinically validated positive detection rate of no less than 90% and a negative accuracy rate of no less than 90%.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention achieves non-invasive and user-friendly sample collection. The exhaled condensate (EBC) collection process is comfortable and non-invasive, suitable for all age groups from children to adults (testing range 4-57 years old), effectively avoiding the discomfort caused by invasive sampling such as nasopharyngeal swabs, and eliminating sampling errors caused by differences in operation or resistance from the subject, significantly improving user compliance and the level of sampling standardization.

[0021] 2. This invention achieves efficient and stable sample collection with excellent sample quality. Based on semiconductor refrigeration technology, this invention enables rapid condensation and stable collection of EBCs without pre-cooling, and is insensitive to environmental temperature and humidity interference. The obtained samples are representative and of high quality, especially suitable for detection in the early stages of infection and the window period when viral load is low, matching the sample requirements of the gold standard detection method.

[0022] 3. This invention provides detection capabilities with high sensitivity and rapid response. Based on a label-free electrochemical sensing mechanism, the sensor achieves detection sensitivity for target pathogens at the fg / mL level and completes signal response within 30 seconds. The entire detection process requires no complex nucleic acid extraction or amplification steps or the addition of exogenous redox reagents, making the operation extremely simple.

[0023] 4. This invention possesses outstanding early diagnostic capabilities. With extremely high detection sensitivity, this invention can effectively identify viral infection even when the infected individual is asymptomatic or when the commercial antigen test result is negative, thereby achieving early warning and buying valuable time for clinical intervention and breaking the transmission chain.

[0024] 5. This invention ensures high accuracy and specificity of the detection results. Clinical sample validation has demonstrated high accuracy. The sensor achieves highly selective binding to the target virus by specifically recognizing receptors (such as ACE2), exhibiting strong resistance to cross-reactivity and effectively reducing the risk of false positives and false negatives.

[0025] 6. This invention adopts a platform-based and modular design, exhibiting excellent scalability. Each module of this invention is easy to maintain, upgrade, and mass-produce. By replacing the biometric elements on the sensor surface, the detection capability can be quickly expanded to other respiratory pathogens, thereby flexibly addressing the detection needs of future emerging and re-emerging infectious diseases.

[0026] 7. This invention is highly integrated and perfectly adapted to home and community screening scenarios. It integrates non-invasive sampling, instant detection, and intelligent analysis and interpretation into one system. The operation process is simple and quick, and the test results are presented intuitively through a smartphone application. The entire system is portable and easy to use, making it ideal for home self-testing, community screening, and on-site testing in primary healthcare institutions, providing a convenient and reliable instant diagnostic tool for the prevention and control of respiratory infectious diseases. Attached Figure Description

[0027] Figure 1 This is a block diagram of a device for collecting exhaled breath condensate in an embodiment of the present invention; Figure 2 This is a flowchart illustrating the rapid collection, electrochemical detection, and result determination of EBC in this embodiment of the invention; Figure 3This is a schematic diagram of the main body of the device for collecting exhaled breath condensate in an embodiment of the present invention; Figure 4 This is a graph showing the stability test results of the semiconductor refrigeration module of the device for collecting exhaled breath condensate in an embodiment of the present invention; Figure 5 This is a diagram illustrating the EBC collection effect and applicability verification of the device for collecting exhaled condensate in an embodiment of the present invention. Figure 6 This is a comparison of the accuracy of qRT-PCR based on EBC samples and label-free electrochemical detection throughout the entire infection cycle in this embodiment of the invention. Figure 7 This is a schematic diagram of the CV and DPV curves of the stepwise modification process of the label-free electrochemical sensor ACE2 / PBA / SWCNT / SPE in an embodiment of the present invention; Figure 8 This is a schematic diagram of the DPV response curve and fitting curve of the label-free electrochemical sensor ACE2 / PBA / SWCNT / SPE to the SARS-CoV-2 spike protein (SP) in an embodiment of the present invention. Figure 9 This is a graph showing the specificity identification and long-term storage stability test results of the ACE2 / PBA / SWCNT / SPE label-free electrochemical sensor in the embodiments of the present invention. Figure 10 This is a schematic diagram comparing the it response sensitivity of the spike protein (SP) and pseudovirus particles (PSV) in an embodiment of the present invention; Figure 11 This is a schematic diagram illustrating the selectivity and multi-target recognition performance in the it mode of this invention embodiment; Figure 12 This is a schematic diagram of the parameter setting interface and EBC sample analysis interface of the fast it curve processing application (APP) in an embodiment of the present invention. Figure 13 This is a schematic diagram illustrating the threshold division of positive and negative sample responses and the differentiation of different strains and symptoms in an embodiment of the present invention. Figure 14 This is a schematic diagram illustrating the monitoring of the entire infection cycle, the universality of multiple samples, and the accuracy of monitoring different symptoms in this embodiment of the invention; Figure 15 This is a flowchart of a home-based respiratory virus testing method in an embodiment of the present invention. Detailed Implementation

[0028] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0029] Example 1 This invention provides a device for collecting exhaled breath condensate. This device integrates semiconductor rapid condensation technology and a label-free electrochemical receptor sensor, making it a home-based testing device. This device enables stable and efficient collection of exhaled breath condensate (EBC) and, through specific and highly sensitive detection of respiratory viruses such as SARS-CoV-2, achieves efficient screening for early-stage, asymptomatic infections. It also has the ability to differentiate between different symptom severity levels, providing a timely and reliable home diagnostic tool for the prevention and control of various respiratory infectious diseases (such as MERS-CoV, SARS-CoV, and H1N1). The exhaled breath condensate collection device of this invention is particularly suitable for early, home-based screening of the novel coronavirus (SARS-CoV-2) and other respiratory pathogens.

[0030] like Figures 1 to 14 As shown, the device for collecting exhaled breath condensate according to an embodiment of the present invention includes: The main body includes a semiconductor cooling module, an airflow channel at least partially disposed inside the semiconductor cooling module, and a disposable unit detachably connected to the inlet of the airflow channel; the airflow channel is formed by a thin tube coiled inside the semiconductor cooling module, used to condense exhaled gas to obtain an exhaled gas condensate sample; the disposable unit includes a mouthpiece and a connecting tube, the mouthpiece being connected to the connecting tube, and the connecting tube being connected to the inlet of the airflow channel; A label-free electrochemical sensor for receiving exhaled condensate samples from the subject is constructed by depositing a carboxylated single-walled carbon nanotube film in the working area of ​​a screen-printed electrode and immobilizing angiotensin-converting enzyme 2 receptor on the film using a 1-pyrene butyrate N-hydroxysuccinimide crosslinking agent. The analysis module is an application program installed on an electronic device. The analysis module has a built-in machine learning algorithm and is communicatively connected to the label-free electrochemical sensor. It is configured to receive the electrical signals from the label-free electrochemical sensor and perform automated analysis, comparison, and result output.

[0031] The apparatus for collecting exhaled breath condensate according to an embodiment of the present invention will be described in further detail below.

[0032] It is understandable that biosensors are often constructed using antibody-based methods, which involve complex modification steps and require the modification of electroactive groups or the provision of electroactive substances to detect signals. In this invention, to improve the sensor's detection sensitivity and performance, carboxylated carbon nanotubes (SWCNTs) and 1-pyrene butyrate N-hydroxysuccinimide ester (PBASE) are used to provide more angiotensin-converting enzyme 2 (ACE2) binding sites, enabling the specific recognition of the SARS-CoV-2 virus via the ACE2 receptor.

[0033] The device for collecting exhaled breath condensate according to embodiments of the present invention addresses the key needs of early, non-invasive, and user-friendly home testing. This device, EBCatch (Exhaled Breath Condensate Analysis for Transmission Control & Health-security), is a non-invasive, rapid, and highly sensitive platform for direct detection and rapid diagnostic output of respiratory viruses. This platform integrates an ergonomically designed exhaled breath condensate collection device, a receptor-based electrochemical sensor, and a machine learning-assisted smartphone application, enabling seamless operation. Figure 2 As shown. The device for collecting exhaled breath condensate in this embodiment of the invention differs from existing mask-based condensers that are susceptible to dilution by environmental vapors and prolonged sampling. This device achieves rapid condensation without pre-cooling, effectively reducing environmental interference and ensuring high-concentration sample enrichment within 1 minute. Furthermore, existing integrated systems often suffer from complex manufacturing processes and exposure risks, while EBCatch employs a modular functional coupling design: maintaining physical isolation between condensation and detection avoids aerosol contamination, while disposable components in both modules ensure operational safety and user comfort. EBCatch's core advantage lies in its ability to achieve early screening during the asymptomatic infection period when antigen detection often fails, thereby significantly reducing the risk of transmission. By replacing biometric elements, the device can easily achieve highly specific multiplex detection of various respiratory pathogens. Combined with a smartphone application, leveraging machine learning to automatically perform signal analysis and result interpretation, EBCatch provides rapid, accurate, and user-friendly diagnostic services in real-time scenarios, enabling timely and accurate implementation of prevention and control strategies for novel pathogens (such as disease X). The EBCatch usage process is as follows: Figure 2As shown, the process includes three key steps: 1. EBC collection: The user gently exhales through a disposable ergonomic mouthpiece connected to a semiconductor cooling module. This embodiment of the invention can efficiently condense respiratory aerosols, typically generating a sufficient EBC sample (≈80 µL) for detection within 30 seconds. This reduces user discomfort and avoids the risk of sample dilution or degradation, effectively overcoming the limitations of traditional mask-type or commercial condenser methods. 2. Label-free electrochemical detection: A small amount of EBC is directly added to an ACE2-functionalized screen-printed electrode (SPE), and real-time detection is achieved using chronoamperometry (it), enabling rapid readings within 30 seconds without reagents. 3. Automated analysis via a dedicated smartphone application: This embodiment of the invention classifies it results according to preset thresholds and outputs diagnostic results (positive / negative / retest) within 30 seconds. The entire process from sample collection to result delivery takes only 8 minutes, making EBCatch a practical solution for screening early asymptomatic infections.

[0034] The overall workflow of the device for collecting exhaled condensate according to this invention is as follows: the user gently exhales into the collection module through the mouth for 30 seconds to 3 minutes → the collected EBC droplets are directly added to the electrochemical sensor → it is detected in PBS (30 seconds) → the mobile app automatically analyzes and outputs the diagnostic results. The entire process can be completed within 8 minutes.

[0035] In this embodiment, the EBC non-invasive stable and efficient collection system (i.e., the main body of the device for collecting exhaled condensate) includes: a semiconductor cooling module, an airflow channel, and a disposable unit.

[0036] Furthermore, the semiconductor cooling module includes an aluminum semiconductor cooling block, a heat insulation pad, a heat sink, and a cooling fan. Utilizing semiconductor cooling technology, it can reduce the condensation surface temperature to -5 to -2 °C within 7-8 minutes of startup without pre-cooling and maintain stability thereafter.

[0037] Furthermore, the airflow channel employs a silicone tube design, coiled inside the semiconductor block, maximizing the contact area to enhance heat exchange efficiency while effectively suppressing interference from ambient temperature and vapor. Optimized airflow paths ensure that even in febrile patients (exhaled air approximately 39 °C), the condenser temperature fluctuation is less than 1.2 °C, guaranteeing collection efficiency and stability. Approximately 80 μL of EBC can be collected within 30 seconds, meeting the needs of immediate testing; approximately 580 μL can be collected within 3 minutes, meeting the requirements for qRT-PCR retesting.

[0038] Furthermore, the disposable unit design includes a detachable food-grade silicone nozzle and connecting hose, forming a disposable airflow path. It can be discarded after a single use, ensuring user comfort while eliminating the risk of cross-contamination.

[0039] Furthermore, after the main body of the device for collecting exhaled breath condensate in this embodiment of the invention is encapsulated with a 3D-printed shell, the final device has a height, width, and depth of 13.7 × 19.6 × 17.4 cm. 3 Weighing 1.15 kg (including a 0.4 kg cooling module and a 0.37 kg external power supply), it achieves portability and is suitable for practical on-the-go care and home testing applications.

[0040] In this embodiment, the construction of the electrochemical acceptor sensor (i.e., the label-free electrochemical sensor) is detailed as follows: Specifically, the construction steps for the ACE2 / PBA / SWCNT / SPE sensor are as follows: Fabrication of the ACE2 / PBA / SWCNT / SPE sensor: First, a 1 mg / mL SWCNT solution was prepared by optimizing the dimethylformamide (DMF) / water solvent ratio (1:3, v / v). This solution was deposited onto the SPE working region through three consecutive drop casting cycles and intermittent drying to ensure a uniform film formation and high electron transport efficiency, thereby achieving sensitive signal amplification. Using organic crosslinking agents including EDC / NHS and PBASE, the SWCNT surface was activated through the π-π stacking of the pyrene groups in PBASE. The ACE2 receptor was then immobilized with the amino groups (-NH2) at the carboxylic acid terminus (-COOH) of SWCNTs and PBASE via a binding reaction. The ACE2 receptor was immobilized by covalently linking it to the activated carboxyl site. Bovine serum albumin (BSA) was used for non-specific site blocking to reduce background interference, ultimately constructing the ACE2 / PBA / SWCNT / SPE biosensor. By immobilizing ACE2 receptors on the electrode surface and binding them to target analytes, rapid detection of the surface spike protein (SP) and viral particles (PSV) of SARS-CoV-2 virus in positive EBC samples can be achieved.

[0041] Specifically, the electrochemical signal response of the label-free electrochemical sensor is achieved through the following two modes: DPV detection mode: In a solution containing potassium ferricyanide, the weakening of the redox peak current before and after the virus is captured by the sensor (signal suppression effect), thereby enabling the quantification of viral proteins.

[0042] Direct detection mode (IT): Direct detection via chronoamperometry (IT) in PBS buffer. When SARS-CoV-2 virus particles or their spike protein (S protein) bind to ACE2 on the electrode surface, a change in the charge distribution at the electrode interface occurs, leading to a change in the current response, without the need for any exogenous redox mediators. By recording the current-time curve generated by the sensor's response to target binding at a constant potential, direct and rapid detection of virus particles is achieved.

[0043] In this embodiment, the specific details of actual sample validation and multi-target expansion are as follows: Verification of the accuracy of actual samples: EBC, saliva and nasal swab samples were collected from suspected infected individuals. Nasal swabs were also collected for antigen test strip testing. The obtained EBC samples were further verified for positive and negative results by qRT-PCR testing.

[0044] Electrochemical response of actual samples: EBC samples were directly dropped onto the surface of the electrochemical sensor for DPV or it detection. The changes in current signal were analyzed to determine whether the target virus was present in the sample, and the results were compared with those of antigen test strips and qRT-PCR.

[0045] Multi-target detection expansion: By replacing the recognition receptors on the electrode surface with specific antibodies / aptamers targeting the MERS-CoV spike protein or SARS-CoV spike protein, specific recognition of the corresponding viruses can be achieved, and an electrochemical sensor array that can be used for screening multiple respiratory viruses can be constructed.

[0046] In this embodiment, the development of a machine learning and data processing app is mainly achieved through the following steps: Random forest selection: Let t0 be the time after the sample is added, I0 be the initial current, I1 be the response current at response time t1, and a random vector and model be used. (I-I0) / I0 are the model parameters. Based on the response results of the sample addition order in the it curve, the maximum value of different response time widths is selected.

[0047] Development of an automated data processing app: Input standard samples, it curves of 81 positive EBC samples and 74 negative EBC samples for COVID-19, use (I-I0) / I0 as the model parameter, and filter for the maximum and average response values ​​within different time ranges. For each threshold range, output the corresponding negative, positive, or retest results.

[0048] To verify the effectiveness of the device for collecting exhaled condensate according to the embodiments of the present invention, the device for collecting exhaled condensate according to the embodiments of the present invention is tested and evaluated through specific experiments.

[0049] 1. The use and performance verification of the EBC collection module (i.e., the main body of the device for collecting exhaled condensate) are as follows: (1) The key components of the main body and their 3D physical images are as follows: Figure 3 As shown. Instructions for use: Connect to power, start the collector, and wait 7-8 minutes for the semiconductor cooling block (i.e., the semiconductor cooling module) to stabilize. The subject gently exhales into the collector at a normal breathing rate through a disposable silicone mouthpiece for 1-3 minutes. After collection, remove the reservoir tube, aliquot the sample and store it at -20 or -80°C, or immediately aspirate approximately 300 μL of EBC sample for nucleic acid extraction and qRT-PCR amplification, and take 1 μL for electrochemical it detection.

[0050] (2) Device stability: The cooling effect was measured after 4 hours of automatic start-up and 4 minutes of continuous exhalation. The temperature fluctuation was small, indicating that the device has good cooling stability.

[0051] (3) Collection effect: The test showed that the EBC volume of volunteers with different age ranges, positive and negative for novel coronavirus was measured after continuous and slow exhalation for 4 minutes. The results showed that the device had a high collection efficiency of 198 µL / min and good age range and universality.

[0052] (4) Sample accuracy and sampling comfort assessment: EBC and nasal swab samples were collected at the infection positive and negative points. The accuracy of the samples was verified by qRT-PCR, which showed that the viral load and activity in the early stage of infection were better than those in nasal swab samples. At the same time, EBC samples were collected for the entire cycle of infection window period, positive point, negative point and 9 days after negative point for qRT-PCR and electrochemical it test. The results showed that the EBC samples obtained by this method have the ability to monitor the entire cycle of infection, and the detection performance for the infection window period and positive point is better than that of antigen test strips. The satisfaction of volunteers with the sampling process was collected. It showed that among the participants, 88% of the operation process was "comfortable", 89% described the 3-minute exhalation process as "easy", and there were very few requests for operation adjustment (e.g., 67% of users reported "no suggestions for improvement").

[0053] Unlike antigen test strips, whose results are easily affected by user-dependent sampling errors (such as insufficient nasal penetration leading to invalid results, insufficient mucosal contact causing false negative readings), the EBC collection method of this invention provides robust and operator-independent reliability by simplifying the collection process. It can detect the entire infection cycle and its accuracy for the infection window period is significantly better than that of antigen test strips.

[0054] 2. Preparation and characterization of label-free electrochemical sensors, with specific experimental steps as follows: (1) Stepwise preparation and characterization of the ACE2 / PBA / SWNT / SPE label-free sensor: The preparation method is as described above, and characterization was performed by CV and DPV tests in 5 mM potassium ferricyanide. In the CV measurement, a pair of distinct redox peaks with different current intensities were observed, indicating that there is a difference in the charge transfer ability of the modified layers. At the same time, the peak current increased after the carboxylated SWCNT deposition, confirming the good conductivity of the carboxylated SWCNT layer. After the SWCNT surface was activated by EDC / NHS and PBASE, the conductivity decreased significantly, while the peak current slightly increased after ACE2 immobilization. After BSA blocking, the peak current of the ACE2 / PBA / SWCNT / SPE label-free sensor was significantly reduced due to BSA acting as a barrier to interfacial electron transfer. Nevertheless, the electrode still maintains sufficient charge transfer efficiency at the solid-liquid interface, which can generate a quantifiable current signal for virus detection. The DPV results are consistent with the data obtained from the CV curves, further verifying the successful stepwise assembly of the functional components on the electrode surface.

[0055] (2) Performance characterization in potassium ferricyanide: The target SP was gradually added to the potassium ferricyanide solution to obtain the DPV response curve. The standard curve was obtained by fitting, and the LOD of the sensor was found to be 0.12 pg / mL.

[0056] (3) Response specificity and stability: ACE2 / PBA / SWNT / SPE has specific target recognition performance, and its stability at 4℃ is better than that at 25℃ and 37℃. It still has more than 75% response activity after long-term storage for 2 months.

[0057] 3. The direct it response performance of the label-free electrochemical sensor ACE2 / PBA / SWCNT / SPE was investigated using the following experimental procedures: (1) Response sensitivity: Take 1 µL of target C at different concentrations SP The iterative testing (it) was performed sequentially, and the results showed that at the same concentration, the response of PSV was much greater than that of SP, which is related to the presence of multiple SP sites on the surface of PSV. The target SP and PSV were tested using the iterative method with sequential addition, and the results showed that PSV had a higher response sensitivity than SP and lower LOD values, at 1.6 fg / mL and 44 pg / mL, respectively.

[0058] (2) Selectivity and multi-target recognition: Different substances were tested sequentially in the test, and the response to SARS-CoV-2 was found to be the most obvious. Although the response to SARS was more obvious than that to potassium ferricyanide, it can be attributed to the highly similar SP structure. However, the possibility of both existing in real life is small, so it does not affect the specific recognition of the target. At the same time, by changing the receptor of the sensor, the specific recognition of multiple targets can be achieved.

[0059] 4. The application of a device for collecting exhaled breath condensate for rapid detection of EBC samples from actual patients infected with the novel coronavirus. The specific experimental steps are as follows: (1) Use of the quick processing program APP (i.e. analysis module): Input the IT test curve into the APP, set the required reading time range, and the average value of (I-I0) / I0 of the sample response will be given within 1 minute, and the result will be automatically given as negative, positive or needing to be retested.

[0060] (2) Detection of positive and negative EBCs for the novel coronavirus: EBC samples were collected after a 3-minute slow exhalation. After verifying the accuracy of the samples by qRT-PCR, it was tested to obtain the (I-I0) / I0 values ​​of 74 negative and 81 positive EBC samples, and the thresholds for negative and positive were obtained. The thresholds were used to distinguish the responses of different types of strains and different symptom stages. The results showed that both the original strain and the variant strain could be identified. This method can be applied to the detection of EBC samples in asymptomatic infection, symptomatic infection, and EBCs that have turned negative.

[0061] (3) Detection of positive and negative EBCs for the novel coronavirus: EBC samples from patients throughout the infection cycle were collected. After verifying the accuracy of the samples by qRT-PCR, it was tested. Based on the above thresholds, EBC samples were tested at 2 days of the infection window period, the day of positive result, 4 days of positive result, the day of negative result, and 2 days after negative result. It was found that the device has the accuracy matching qRT-PCR, and the sensitivity at the window period and positive point is much higher than that of antigen test strips, showing better application prospects in home self-testing.

[0062] The developed device was found to have good sample universality when collecting and testing EBC, nasal swabs, and saliva samples from individuals at the same stage of infection. The device was then used to validate the accuracy of EBC sample testing for negative and positive results and symptom differentiation. Comparison with the gold standard qRT-PCR showed a positive detection rate of 95.1%, a negative accuracy of 97.3%, and a symptom differentiation accuracy as high as 93.8%.

[0063] Example 2 like Figure 15 As shown, this embodiment of the invention also provides a home-based method for detecting respiratory viruses using a device for collecting exhaled condensate as described in Embodiment 1, comprising the following steps: Sample collection steps: The user exhales into the device for collecting exhaled condensate through the disposable unit for 30 seconds to 5 minutes to obtain an exhaled condensate sample; Direct detection step: Take no more than 5 µL of the exhaled breath condensate sample and drop it directly onto the electrode surface of the label-free electrochemical sensor. Detect the current-time curve and obtain the signal value (I-I0) / I0 within 60 seconds using chronoamperometry in phosphate buffer. Intelligent interpretation steps: The signal value is input into the analysis module, which is automatically analyzed by the machine learning algorithm built into the analysis module, and outputs a positive, negative or retesting result within 60 seconds.

[0064] Furthermore, during the sample collection step, the user exhales gently at a normal breathing rate.

[0065] Furthermore, the entire process of this home-based respiratory virus testing method, from sample collection to result output, takes less than 8 minutes.

[0066] Furthermore, this home-based respiratory virus testing method can effectively detect samples during the infection window period, asymptomatic infection period, and antigen-negative samples, with a clinically validated positive detection rate of no less than 90% and a negative accuracy rate of no less than 90%.

[0067] The above is a detailed description of the preferred embodiments of the present invention. However, the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention. All such equivalent modifications or substitutions are included within the scope defined by the claims of this application.

Claims

1. A device for collecting exhaled breath condensate, characterized in that, include: The main body includes a semiconductor cooling module, an airflow channel at least partially disposed inside the semiconductor cooling module, and a disposable unit detachably connected to the inlet of the airflow channel; the airflow channel is formed by a thin tube coiled inside the semiconductor cooling module, used to condense exhaled gas to obtain an exhaled gas condensate sample; the disposable unit includes a mouthpiece and a connecting tube, the mouthpiece being connected to the connecting tube, and the connecting tube being connected to the inlet of the airflow channel; A label-free electrochemical sensor for receiving exhaled condensate samples from the subject is constructed by depositing a carboxylated single-walled carbon nanotube film in the working area of ​​a screen-printed electrode and immobilizing angiotensin-converting enzyme 2 receptor on the film using a 1-pyrene butyrate N-hydroxysuccinimide crosslinking agent. The analysis module is an application program installed on an electronic device. The analysis module has a built-in machine learning algorithm and is communicatively connected to the label-free electrochemical sensor. It is configured to receive the electrical signals from the label-free electrochemical sensor and perform automated analysis, comparison, and result output.

2. The device for collecting exhaled breath condensate according to claim 1, characterized in that, The length of the main body is no more than 25 cm, the width is no more than 20 cm, the height is no more than 20 cm, and the weight is no more than 2 kg.

3. The device for collecting exhaled breath condensate according to claim 1, characterized in that, The design of the airflow channel ensures that the temperature fluctuation of its condensation area is less than 2°C under the exhaled airflow conditions of a febrile patient.

4. The device for collecting exhaled breath condensate according to claim 1, characterized in that, The main body is configured to collect at least 50 µL of exhaled condensate within 30 seconds of continuous, gentle exhalation by the user; and at least 500 µL of exhaled condensate within 5 minutes of exhalation.

5. The device for collecting exhaled breath condensate according to claim 1, characterized in that, The label-free electrochemical sensor has a detection limit of no more than 10 fg / mL for SARS-CoV-2 pseudovirus particles.

6. The device for collecting exhaled breath condensate according to claim 1, characterized in that, The analysis module is configured to use the rate of change of current response value (I-I0) / I0 as the analysis parameter, where I0 is the initial current value before the sample is added and I is the response current value at a specific time after the sample is added; and to compare this parameter with a threshold predetermined by clinical samples.

7. A method for home detection of respiratory viruses using a device for collecting exhaled breath condensate as described in any one of claims 1 to 6, characterized in that, Includes the following steps: Sample collection steps: The user exhales into the device for collecting exhaled condensate through the disposable unit for 30 seconds to 5 minutes to obtain an exhaled condensate sample; Direct detection step: Take no more than 5 µL of the exhaled breath condensate sample and drop it directly onto the electrode surface of the label-free electrochemical sensor. Detect the current-time curve and obtain the signal value (I-I0) / I0 within 60 seconds using chronoamperometry in phosphate buffer. Intelligent interpretation steps: The signal value is input into the analysis module, which is automatically analyzed by the machine learning algorithm built into the analysis module, and outputs a positive, negative or retesting result within 60 seconds.

8. The home-based respiratory virus testing method according to claim 7, characterized in that, During the sample collection process, the user exhales gently at a normal breathing rate.

9. The home-based respiratory virus testing method according to claim 7, characterized in that, The entire process of this home-based respiratory virus testing method, from sample collection to result output, takes less than 8 minutes.

10. The home-based respiratory virus testing method according to claim 7, characterized in that, This home-based respiratory virus testing method can effectively detect samples during the window period of infection, the asymptomatic infection period, and those that are negative for antigen testing. Its clinically validated positive detection rate is no less than 90%, and its negative accuracy rate is no less than 90%.

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