A Wax Interface-Enabled One-Pot Multiplexed Nucleic Acid Testing Platform for Rapid and Sensitive Detection of Viruses and Variants.

High-quality, low-cost, and rapid detection is essential for the society to reopen the economy during the critical period of transition from Coronavirus Disease 2019 (COVID-19) pandemic response to pandemic control. In addition to performing sustainable and target-driven tracking of SARS-CoV-2, conducting comprehensive surveillance of variants and multiple respiratory pathogens is also critical due to the frequency of reinfections, mutation immune escape, and the growing prevalence of the cocirculation of multiple viruses. By utilizing a 0.05 cents wax interface, a Stable Interface assisted Multiplex Pathogenesis Locating Estimation in Onepot (SIMPLEone) using nested RPA and CRISPR/Cas12a enzymatic reporting system is successfully developed. This smartphone-based SIMPLEone system achieves highly sensitive one-pot detection of SARS-CoV-2 and its variants, or multiple respiratory viruses, in 40 min. A total of 89 clinical samples, 14 environmental samples, and 20 cat swab samples are analyzed by SIMPLEone, demonstrating its excellent sensitivity (3-6 copies/reaction for non-extraction detection of swab and 100-150 copies/mL for RNA extraction-based assay), accuracy (>97.7%), and specificity (100%). Furthermore, a high percentage (44.2%) of co-infection cases are detected in SARS-CoV-2-infected patients using SIMPLEone's multiplex detection capability.

Using Microfluidic Chip and Allele-Specific PCR to Rapidly Identify Drug Resistance-Associated Mutations of Mycobacterium tuberculosis.

Background: The currently used conventional susceptibility testing for drug-resistant Mycobacterium tuberculosis (M.TB) is limited due to being time-consuming and having low efficiency. Herein, we propose the use of a microfluidic-based method to rapidly detect drug-resistant gene mutations using Kompetitive Allele-Specific PCR (KASP). Methods: A total of 300 clinical samples were collected, and DNA extraction was performed using the "isoChip®" Mycobacterium detection kit. Phenotypic susceptibility testing and Sanger sequencing were performed to sequence the PCR products. Allele-specific primers targeting 37 gene mutation sites were designed, and a microfluidic chip (KASP) was constructed using 112 reaction chambers to simultaneously detect multiple mutations. Chip validation was performed using clinical samples. Results: Phenotypic susceptibility of clinical isolates revealed 38 rifampicin (RIF)-resistant, 64 isoniazid (INH)-resistant, 48 streptomycin (SM)-resistant and 23 ethambutol (EMB)-resistant strains, as well as 33 multi-drug-resistant TB (MDR-TB) strains and 20 strains fully resistant to all four drugs. Optimization of the chip-based detection system for drug resistance detection showed satisfactory specificity and maximum fluorescence at a DNA concentration of 1×101 copies/µL. Further analysis revealed that 76.32% of the RIF-resistant strains harbored rpoB gene mutations (sensitivity, 76.32%; specificity 100%), 60.93% of the INH-resistant strains had katG gene mutations (sensitivity, 60.93%; specificity, 100%), 66.66% of the SM-resistant strains carried drug resistance gene mutations (sensitivity, 66.66%; specificity, 99.2%), and 69.56% of the EMB-resistant strains had embB gene mutations (sensitivity, 69.56%; specificity, 100%). Further, the overall agreement between the microfluidic chip and Sanger sequencing was satisfactory, with a turnaround time of the microfluidic chip was approximately 2 hours, much shorter than the conventional DST method. Conclusion: The proposed microfluidic-based KASP assay provides a cost-effective and convenient method for detecting mutations associated with drug resistance in M. tuberculosis. It represents a promising alternative to the traditional DST method, with satisfactory sensitivity and specificity and a much shorter turnaround time.

Comparison of Three Lateral Flow Immunoassay Formats for the Detection of Antibodies against the SARS-CoV-2 Antigen.

Reliable detection of specific antibodies against pathogens by lateral flow immunoassay (LFIA) greatly depends on the composition of the detectable complex and the order of its assembly. We compared three LFIA formats for revealing anti-SARS-CoV-2 antibodies in sera with the following detected complexes in the analytical zone of the strip: antigen-antibodies-labeled immunoglobulin-binding protein (Scheme A); antigen-antibodies-labeled antigen (Scheme B); and immunoglobulin-binding protein-antibodies-labeled antigen (Scheme C). The lowest detection limit was observed for Scheme C, and was equal to 10 ng/mL of specific humanized monoclonal antibodies. When working with pooled positive sera, Scheme C had a detection limit 15 times lower than Scheme B and 255 times lower than Scheme A. Due to the high sensitivity of Scheme C, its application for the panel of human sera (n = 22) demonstrated 100% diagnostic specificity and sensitivity. These consistent results be useful for designing the format of LFIA serodiagnosis for other diseases.

SMART: A Swing-Assisted Multiplexed Analyzer for Point-of-Care Respiratory Tract Infection Testing.

Respiratory tract infections such as the ongoing coronavirus disease 2019 (COVID-19) has seriously threatened public health in the last decades. The experience of fighting against the epidemic highlights the importance of user-friendly and accessible point-of-care systems for nucleic acid (NA) detection. To realize low-cost and multiplexed point-of-care NA detection, a swing-assisted multiplexed analyzer for point-of-care respiratory tract infection testing (SMART) was proposed to detect multiple respiratory tract pathogens using visible loop-mediated isothermal amplification. By performing hand-swing movements to generate acceleration force to distribute samples into reaction chambers, the design of the SMART system was greatly simplified. By using different format of chips and integrating into a suitcase, this system can be applied to on-site multitarget and multi-sample testing. Three targets including the N and Orf genes of SARS-CoV-2 and the internal control were simultaneously analyzed (limit of detection: 2000 copies/mL for raw sample; 200 copies/mL for extracted sample). Twenty-three clinical samples with eight types of respiratory bacteria and twelve COVID-19 clinical samples were successfully detected. These results indicate that the SMART system has the potential to be further developed as a versatile tool in the diagnosis of respiratory tract infection.

Silent Antibodies Start Talking: Enhanced Lateral Flow Serodiagnosis with Two-Stage Incorporation of Labels into Immune Complexes.

The presence of pathogen-specific antibodies in the blood is widely controlled by a serodiagnostic technique based on the lateral flow immunoassay (LFIA). However, its common one-stage format with an antigen immobilized in the binding zone of a test strip and a nanodispersed label conjugated with immunoglobulin-binding proteins is associated with risks of very low analytical signals. In this study, the first stage of the immunochromatographic serodiagnosis was carried out in its traditional format using a conjugate of gold nanoparticles with staphylococcal immunoglobulin-binding protein A and an antigen immobilized on a working membrane. At the second stage, a labeled immunoglobulin-binding protein was added, which enhanced the coloration of the bound immune complexes. The use of two separated steps, binding of specific antibodies, and further coloration of the formed complexes, allowed for a significant reduction of the influence of non-specific immunoglobulins on the assay results. The proposed approach was applied for the serodiagnosis using a recombinant RBD protein of SARS-CoV-2. As a result, an increase in the intensity of test zone coloration by more than two orders of magnitude was demonstrated, which enabled the significant reduction of false-negative results. The diagnostic sensitivity of the LFIA was 62.5% for the common format and 100% for the enhanced format. Moreover, the diagnostic specificity of both variants was 100%.

Sensitive and Rapid Diagnosis of Respiratory Virus Coinfection Using a Microfluidic Chip-Powered CRISPR/Cas12a System.

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 is profoundly influencing the global healthcare system and people's daily lives. The high resource consumption of coronavirus disease 2019 (COVID-19) is resulting in insufficient surveillance of coinfection or resurgence of other critical respiratory epidemics, which is of public concern. To facilitate evaluation of the current coinfection situation, a microfluidic system (MAPnavi) is developed for the rapid (<40 min) and sensitive diagnosis of multiple respiratory viruses from swab samples in a fully sealed and automated manner, in which a nested-recombinase polymerase amplification and the CRISPR-based amplification system is first proposed to ensure the sensitivity and specificity. This novel system has a remarkably low limit of detection (50-200 copies mL-1 ) and is successfully applied to detect 171 clinical samples (98.5% positive predictive agreement; 100% negative predictive agreement), and the results identify 45.6% coinfection among clinical samples from patients with COVID-19. This approach has the potential to shift diagnostic and surveillance efforts from targeted testing for a high-priority virus to comprehensive testing of multiple virus sets and to greatly benefit the implementation of decentralized testing.

A fully integrated SNP genotyping system for hereditary hearing-loss detection.

Hereditary hearing loss is one of the most common human neurosensory disorders, and there is a great need for early intervention methods such as genetically screening newborns. Single nucleotide polymorphisms (SNPs) are the major genetic targets for hearing-loss screening. In this study, a fully integrated SNP genotyping system was constructed to identify hereditary hearing loss-related genetic markers from human whole blood. The entire detection process, including blood cell lysis, nucleic acid extraction, the reaction mixture distribution, the chambers sealing and the two-colour multiplex competitive allele-specific polymerase chain reaction (KASP), can be automatically conducted in a self-contained cassette within 3 hours. To critically evaluate the performance of the system, its specificity, sensitivity and stability were assessed. Then, 13 clinical samples were genotyped with this fluidic cassette system to detect seven hotspot deafness-associated mutations in three genes (MT-RNR1, GJB2 and SLC26A4). The detection results of the cassette system were 100% concordant with those obtained by Sanger sequencing, proving its accuracy in the genetic screening of inherited hearing loss.

Multiplexed detection of respiratory pathogens with a portable analyzer in a "raw-sample-in and answer-out" manner.

Coronavirus disease 2019 (COVID-19) has emerged, rapidly spread and caused significant morbidity and mortality worldwide. There is an urgent public health need for rapid, sensitive, specific, and on-site diagnostic tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this study, a fully integrated and portable analyzer was developed to detect SARS-CoV-2 from swab samples based on solid-phase nucleic acid extraction and reverse transcription loop-mediated isothermal amplification (RT-LAMP). The swab can be directly inserted into a cassette for multiplexed detection of respiratory pathogens without pre-preparation. The overall detection process, including swab rinsing, magnetic bead-based nucleic acid extraction, and 8-plex real-time RT-LAMP, can be automatically performed in the cassette within 80 min. The functionality of the cassette was validated by detecting the presence of a SARS-CoV-2 pseudovirus and three other respiratory pathogens, i.e., Klebsiella pneumoniae, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. The limit of detection (LoD) for the SARS-CoV-2 pseudovirus was 2.5 copies/µL with both primer sets (N gene and ORF1ab gene), and the three bacterial species were successfully detected with an LoD of 2.5 colony-forming units (CFU)/µL in 800 µL of swab rinse. Thus, the analyzer developed in this study has the potential to rapidly detect SARS-CoV-2 and other respiratory pathogens on site in a "raw-sample-in and answer-out" manner.

A Portable Microfluidic System for Point-of-Care Detection of Multiple Protein Biomarkers.

Protein biomarkers are indicators of many diseases and are commonly used for disease diagnosis and prognosis prediction in the clinic. The urgent need for point-of-care (POC) detection of protein biomarkers has promoted the development of automated and fully sealed immunoassay platforms. In this study, a portable microfluidic system was established for the POC detection of multiple protein biomarkers by combining a protein microarray for a multiplex immunoassay and a microfluidic cassette for reagent storage and liquid manipulation. The entire procedure for the immunoassay was automatically conducted, which included the antibody-antigen reaction, washing and detection. Alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and carcinoma antigen 125 (CA125) were simultaneously detected in this system within 40 min with limits of detection of 0.303 ng/mL, 1.870 ng/mL, and 18.617 U/mL, respectively. Five clinical samples were collected and tested, and the results show good correlations compared to those measured by the commercial instrument in the hospital. The immunoassay cassette system can function as a versatile platform for the rapid and sensitive multiplexed detection of biomarkers; therefore, it has great potential for POC diagnostics.

An enhanced centrifugation-assisted lateral flow immunoassay for the point-of-care detection of protein biomarkers.

Protein biomarkers are widely used for disease diagnosis, but the current detection methods utilized in centralized laboratories are mainly based on enzyme-linked immunosorbent assay (ELISA)-derived sandwich-type immunoassays such as chemiluminescent or electrochemiluminescent immunoassays, which suffer from long detection times and cumbersome instruments. For the point-of-care (POC) detection of protein biomarkers, various test strips for lateral flow immunoassay (LFIA) have been manufactured, but their detection sensitivities and capabilities for raw samples are limited. In this study, an enhanced centrifugation-assisted lateral flow immunoassay (ECLFIA) was established to rapidly detect protein biomarkers in whole blood with a higher sensitivity than LFIA. By inserting a nitrocellulose membrane into a centrifugal disc, fully automated operations, including sample preparation, active lateral flow actuation, washing, and signal amplification, which could hardly be performed in conventional LFIA, were enabled on the centrifugal platform for ECLFIA. The entire process for detecting human prostate specific antigen (PSA) in a drop of blood (20 µL) could be completed in 15 min. The limit of detection for our ECLFIA system was 0.028 ng mL-1, showing a 21.4-fold improvement compared to that of LFIA. Moreover, this system was utilized to detect PSA in 34 clinical samples. The results were compared to those measured using a commercial instrument used in the hospital, and a good correlation coefficient of 0.986 was obtained, demonstrating the practicality of this ECLFIA system. In summary, the ECLFIA system established in this study can be an efficient tool for the POC detection of protein biomarkers with comprehensive advantages in sensitivity, simplicity and speed.

A self-contained and fully integrated fluidic cassette system for multiplex nucleic acid detection of bacteriuria.

The gold standard for diagnosing infectious diseases is culture-based identification of bacterial pathogens, which is time-consuming and labour-intensive. Current advances in molecular diagnostics and microfluidic technologies have made the rapid detection of bacteria or viruses in clinical specimens possible. However, the need for rapid, sensitive and multiplex detection of pathogens in a "sample-in and answer-out" manner has not been fully satisfied. In this study, a self-contained and fully integrated fluidic cassette and its supporting analyser were constructed for multiplex detection of bacteria to accelerate the diagnosis of urinary tract infections (UTIs). The fully integrated cassette contains all the necessary components and reagents for bacterial analysis. All of the bacterial analysis processes, including bacterial lysis, magnetic silica bead-based DNA extraction, DNA elution and multiplex loop-mediated amplification (LAMP), are automatically conducted in the cassette. This cassette was successfully applied for the detection of four major pathogenic bacteria in UTIs, i.e., Escherichia coli, Proteus mirabilis, Salmonella typhimurium and Staphylococcus aureus. The first three were successfully detected with a limit of detection (LoD) of 1 colony-forming unit (CFU) µL-1 and the last was with a LoD of 10 CFU µL-1 in urine samples, demonstrating that the cassette has similar sensitivity compared to that of the manual protocol, which is lower than that required by UTIs. The turnaround time for this cassette-based sample-to-answer system was approximately 100 minutes, and the detection is sensitive, fully automated, and accurate, demonstrating the potential to be a useful diagnostic tool for UTIs.

Highly uniform in-situ cell electrotransfection of adherent cultures using grouped interdigitated electrodes.

Cell electrotransfection is an effective approach for transferring exogenous molecules into living cells by electric stimulation. The existing in-situ electrotransfection micro-devices for adherent cells exhibit the drawbacks of low transfection efficiency and low cell viability. An important reason for these drawbacks is the unequal exposure of cells to the electric field. It was found that cells growing directly below the energized electrodes experience a much lower electric field intensity when compared to the cells growing below the spacing area of the electrodes, resulting in low transfection with a strip-like pattern. Therefore, a new strategy for the in-situ electrotransfection of adherent cells growing in a standard 12-well plate is proposed in this study. By sequentially energizing electrodes arranged in a nested and non-contact manner, the cells were exposed to an overall equal intensity of the electric field, and thus a higher efficiency of transfection was achieved. The seven cell lines transfected using this method exhibited high transfection efficiency and high cell viability, demonstrating the potential for studying gene function.

Construction of a rapid microfluidic-based SNP genotyping (MSG) chip for ancestry inference.

In forensics, ancestry inference can provide leads for criminal investigation. Therefore, the time needed to generate results is the first priority. Single nucleotide polymorphisms (SNPs) are widely used genetic markers for ancestry inference. In this study, a rapid microfluidic-based SNP genotyping (MSG) chip was established to detect 72 autosomal SNPs for ancestry inference of East Asian populations. The DNA template was infused into the chip and distributed into reaction chambers with pre-spotted primer pairs under centrifugation. After sealing the inlets/outlets and the connections among adjacent reaction chambers, the chip was placed onto a plate thermocycler for competitive allele-specific PCR. The SNP genotyping results were generated by analyzing the extracted fluorescence signal of each reaction chamber after PCR was completed. The detection process took less time (2.5 h) and was simpler than other SNP genotyping methods, such as SNaPshot and MassArray. To assess the performance of the chip, its accuracy, specificity, and sensitivity were evaluated. A total of 50 samples were genotyped using the chip, and the consistency of all of the typing and sequencing results was 100%. For ancestry inference of unknown individuals, a reference database of 3628 individuals from 57 populations was employed, and the ancestry origin of 110 test samples was inferred, demonstrating that the differentiation of East Asian subpopulations can be achieved through the MSG chip method. Overall, the MSG chip method established in this study can effectively be used for rapid and accurate ancestry inference.

Rapid and Automated Detection of Six Contaminants in Milk Using a Centrifugal Microfluidic Platform with Two Rotation Axes.

Antibiotic residues and illegal additives are among the most common contaminants in milk and other dairy products, and they have become essential public health concerns. To ensure the safety of milk, rapid and convenient screening methods are highly desired. Here, we integrated microarray technology into a microfluidic device to achieve rapid, sensitive, and fully automated detection of chloramphenicol, tetracyclines, enrofloxacin, cephalexin, sulfonamides, and melamine in milk on a centrifugal microfluidic platform with two rotation axes. All the liquid reagent for the immunoassay was prestored in the reagent chambers of the microdevice and can be released on demand. The whole detection can be automatically accomplished within 17 min, and the limits of detection were defined as 0.92, 1.01, 1.83, 1.14, 1.96, and 7.80 µg/kg for chloramphenicol, tetracycline (a typical drug of tetracyclines), enrofloxacin, cephalexin, sulfadiazine (a typical drug of sulfonamides), and melamine, respectively, satisfying the national standards for maximum residue limits in China. Raw milk samples were used to test the performance of the current immunoassay system, and the recovery rates in the repeatability tests ranged from 80 to 111%, showing a good performance. In summary, the immunoassay system established in this study can simultaneously detect six contaminants of four samples in a fully automated, cost-effective, and easy-to-use manner and thus has great promise as a screening tool for food safety testing.

A novel electromagnet-triggered pillar valve and its application in immunoassay on a centrifugal platform.

The lab-on-a-disc is a powerful microfluidic platform that skillfully takes advantage of centrifugal force to controllably drive liquids with the assistance of passive or active valves. However, the passive valves are mainly triggered by the rotation speed and can be easily influenced by the surface chemistry of the channel, while the active valves usually require a complicated fabrication or actuation procedure. In this study, a novel active valve that can be easily triggered by an electromagnet was proposed and applied on the centrifugation platform. This valve, named the electromagnet-triggered pillar (ETP) valve, consisted of a metal pin and pressure sensitive adhesive (PSA) tape, and is closed until the pin is lifted up by an electromagnet to partially separate the PSA tape from the substrate. As a typical application, this valve is utilized to construct a centrifugal chip for mycotoxin detection. With four ETP valves in a unit, the sample and liquid reagents can be sequentially released into the reaction chamber that was spotted with mycotoxin conjugates to accomplish the whole immunoassay. Four mycotoxins (aflatoxin B1, ochratoxin A, T-2 toxin, and zearalenone) were simultaneously detected on this chip with limits of detection lower than the permissible limits set by the regulatory agencies of China, demonstrating the practicability of this easy-to-use active valve.

Enhancing the Sensitivity of Lateral Flow Immunoassay by Centrifugation-Assisted Flow Control.

Lateral flow immunoassay (LFIA) is widely used but is limited by its sensitivity. In this study, a novel centrifugation-assisted lateral flow immunoassay (CLFIA) was proposed that had enhanced sensitivity compared to traditional LFIA based on test strips. For CLFIA, a vaulted piece of nitrocellulose membrane was prepared and inserted into a centrifugal disc. Powered by the centrifugal force, the sample volume on the disc was not limited and the flow rate of the reaction fluid was steady and adjustable at different rotation speeds. It was found that lower rotation speeds and larger sample volumes resulted in greater signal intensity in the nitrocellulose membrane as well as higher sensitivity, indicating that the actively controlled flow on the disc allowed for sensitivity enhancement of CLFIA. To operate CLFIA on the centrifugal disc, a portable and cost-effective operating device was constructed to rotate the disc with a stepper motor and collect the results with a smartphone. The proposed method was successfully applied to detect prostate specific antigen (PSA) in human serum. Standard curves were established for CLFIA and LFIA, and both had correlation coefficients of up to 0.99. Under optimal conditions (1500 rpm rotation speed, 120 µL sample volume), the detection limit of CLFIA reached 0.067 ng/mL, showing a 6.2-fold improvement in sensitivity compared to that of LFIA. With clinical serum samples, a good correlation was observed between PSA concentrations measured by CLFIA and by a bulky commercial instrument in hospital. In summary, this portable, cost-effective, and easy-to-use system holds great promise for biomarker detection with enhanced sensitivity compared to traditional LFIA.

A Microfluidic-Based SNP Genotyping Method for Hereditary Hearing-Loss Detection.

The genotyping of SNPs (single nucleotide polymorphisms) is a prerequisite for the analysis of many genetic diseases, including hereditary hearing-loss. However, the existing methods for SNP detection suffer from a long detection period, tedious operation, and a high risk of carryover contamination. To address these challenges, a microfluidic chip is constructed for rapid and efficient SNP genotyping by dividing the sample into many independent chambers for Kompetitive Allele Specific PCR in this study. Using this strategy, multiple detection can be easily accomplished and the challenge for the establishment of multiplex PCR is fundamentally overcome. The entire detection can be finished within 2 h in a fully sealed manner with this method, which is quite simple compared to SNaPshot and MassArray. After assessment of the basic performance, this chip was applied to screen 15 mutations, including SNPs and InDels (insertion-deletion markers), that can cover more than 80% of cases of hereditary hearing-loss in China. Over 40 clinical samples were analyzed with this microfluidic chip for SNP genotyping, and the results are consistent with that obtained by Sanger sequencing, demonstrating its practicability and potential in the application of genetic disease detection.

Rapid and efficient isolation and detection of extracellular vesicles from plasma for lung cancer diagnosis.

Extracellular vesicles (EVs) are cell-derived nanoscale vesicles that provide promising biomarkers for the non-invasive diagnosis of cancer because they carry important cancer-related DNA, RNA and protein biomarkers. However, the clinical application of EVs is limited by tedious and non-standardized isolation methods that require bulky instrumentation. Here, we propose an easy-to-operate, simple dielectrophoretic (DEP) method for EV isolation with higher recovery efficiency (>83%) and higher purity than ultracentrifugation (UC). The DEP chip reduces the isolation procedure from 8 h to 30 min. To facilitate subsequent analysis, our DEP chip achieved integration of EV isolation and in situ lysis of EVs for the first time. Our chip also achieved on-chip siRNA delivery to EVs isolated by DEP. We found that EVs isolated from the plasma of lung cancer patients contained higher levels of miR-21, miR-191 and miR-192 compared to those from healthy people. With on-chip detection, EGFR in EVs could distinguish lung cancer patients from healthy people. Overall, this study provides an efficient and practical approach to the isolation and detection of EVs, which could be used for the early diagnosis of lung cancer.

Isolation and Visible Detection of Tumor-Derived Exosomes from Plasma.

Exosomes are nanosized extracellular vesicles (ranging from 30 to 120 nm) released from many cells that provide promising biomarkers for the noninvasive diagnosis of cancer. However, traditional exosome-isolation methods are tedious, nonstandardized, and require bulky instrumentation, thus limiting its clinical applications. In this paper, an anion-exchange (AE)-based isolation method was first proposed to isolate exosomes directly from plasma and cell-culture medium with AE magnetic beads within 30 min. Exosomes isolated with AE magnetic beads had higher recovery efficiency (>90%) and less protein impurities than those isolated by ultracentrifugation (UC). Prostate-cancer (PCa) exosomes in plasma were detected in a visual, label-free, and quantitative manner with aptamer-capped Fe3O4 nanoparticles for the first time. The linear range of PCa exosomes was estimated from 0.4 × 108 to 6.0 × 108 particles/mL with a detection limit of 3.58 × 106 particles/mL. The present study provides an efficient and practical approach for the rapid isolation and visible detection of exosomes, which is promising for the early diagnosis of PCa.