Microfluidic Platform for Direct Capture and Analysis of Airborne

Article pubs.acs.org/ac

Microfluidic Platform for Direct Capture and Analysis of Airborne Mycobacterium tuberculosis Wenwen Jing,†,‡,∥ Xiran Jiang,†,∥ Wang Zhao,† Sixiu Liu,† Xunjia Cheng,‡,§ and Guodong Sui*,†,§ †

Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention (LAP3), Department of Environmental Science & Engineering, Fudan University, 220 Handan Road, Shanghai, 200433, P.R. China ‡ Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, P.R. China § Institute of Biomedical Science, Fudan University, Shanghai, 200433, P.R. China ABSTRACT: Airborne Mycobacterium tuberculosis is the main source of tuberculosis infection, which is known as one of the worldwide infectious diseases. Direct capture and analysis of airborne Mycobacterium tuberculosis is essential for disease prevention and control. At present, low concentration of pathogens directly collected from the air is the major drawback for rapid analysis. Herein an integrated microfluidic system capable of airborne Mycobacterium tuberculosis capture, enrichment, and rapid bacteriological immunoassay was developed. The whole detection time was decreased to less than 50 min including 20 min of enrichment and 30 min of immunoreaction analysis. It had the advantages of low detection limit, fast detection speed, and low reagent consumption compared with conventional techniques, showing the potential to become a new airborne pathogen analysis platform.

T

tuberculosis, elution volumes were apparently too large to give enough bacteria concentration for direct molecular diagnosis. Therefore, culturing is always the necessary step for the accurate diagnosis of the Mycobacterium tuberculosis. Additionally, 28 days were usually required for Mycobacterium tuberculosis cultivation on Lowenstein-Jenson medium, which was too long to meet the demand of rapid detection as well as disease early warning. So it was necessary to develop a system capable for airborne Mycobacterium tuberculosis direct capture and analysis. Microfluidics is a rising technique first developed in early 1990s. It combined multiple operating units flexibly and showed unique advantages of integration and miniaturization. All the chambers and valves can be integrated to precisely perform complicated operations. Different channels for various analyses can be fabricated as closed chamber to prevent cross contaminations. It also has the advantages of little sample consumption, high analysis speed, and improved sensitivity compared with conventional analytical techniques, so microfluidic technology was suitable for rapid detection of pathogens.11−14 The research of using microfluidic chip to detect pathogens is getting more and more recognition.15−19 Immunoassay was one of the most important biological analytic methods that were widely used in clinical diagnosis and biological analyses. It showed a unique superiority in sample monitoring with high sensitivity and strong particularity. Microfluidic immunoassay integrated the advantages of these

uberculosis (TB) had become one of the most harmful infectious diseases in recent years.1 In 2010, there were 8.8 million new cases of TB and 1.7 million associated deaths.2 Compared with other diseases, tuberculosis is difficult in prevention and control mainly because it was transported through air so that it can spread rapidly and widely especially in highly populated urban areas. It is a serious threat to the public health and had inevitably caused huge economic losses. At present, detection of the Mycobacterium tuberculosis still relies on the patient serum microscopic diagnosis and limited molecular identification from patient serum samples. It is especially difficult to trace the infectious source by a widely applied method, since it is passive and far behind the initial disease spreading pattern. Direct capture and analysis of airborne Mycobacterium tuberculosis from practical air samples is essential for the disease prevention and control. By integrating pathogen capture, enrichment, and detection steps, it could greatly shorten the analysis time and report the airborne pathogen promptly. Unfortunately, at present there is almost no effective and rapid direct analysis method for direct detection of airborne Mycobacterium tuberculosis due to their low concentrations in air samples. Before being analyzed by some traditional methods including enzyme-linked immunosorbent assay (ELISA),3−5 chemical luminescence immunity analysis, and immune colloidal gold technique, the airborne pathogens usually need capture enrichment and cultivating processes.6−10 As known, the detection limit of traditional methods mentioned above was about 103−105 cfu mL−1, but the elution volume of the traditional bacteria enrichment device was usually at the milliliter level. For airborne Mycobacterium © 2014 American Chemical Society

Received: February 11, 2014 Accepted: May 12, 2014 Published: May 23, 2014 5815

dx.doi.org/10.1021/ac500578h | Anal. Chem. 2014, 86, 5815−5821

Analytical Chemistry

Article

Figure 1. (A) Image of the system for airborne bacteria rapid enrichment and bacteriological diagnosis: (a) airborne bacteria enrichment microfluidic chip, (b) immunoassay microfluidic chip. (B) Schematic illustration and detailed structure of enrichment microfluidic chip. (C) Image of the microfluidic immunoassay chip. The various channels had been loaded with food dyes to visualize the structure of the microfluidic chip. (D,E) Schematic illustrations of designed immunoassay microfluidic chip. Valves and columns were illustrated by different colors: (i) fluidic layer, (ii) control layer. (F) Enlarged diagram showing detailed structure of microfluidic immunoassay chip.

■

EXPERIMENTAL SECTION Chemicals and Reagents. Photoresist (SU-8 2025) was purchased from Microchem. Photoresist (AZ 50XT) was purchased from AZ Electronic Materials USA Corp. Polydimethylsiloxane (PDMS) (RTV-615-044) was purchased from Momentive Specialty Chemicals (NY). Microspheres (Protein A) were purchased from Bang’s Laboratory. BCG (the attenuated strain of Mycobacterium tuberculosis, used in this experiment, Mycobacterium tuberculosis antigen protein Ag85B contained), antigen protein Ag85B, and Monoclonal antibody of Ag85B were purchased from Shanghai Public Health Clinical Center. Rabbit polyclonal of Ag85B and FITC Anti-Mouse secondary antibody were purchased from Abcam (Hong Kong) Ltd. Bovine serum albumin (BSA), Tween20, sodium azide (NaN3), and sodium chloride (NaCl) were purchased from Genebase Gene-Tech Co. (Shanghai, China). Escherichia coli was purchased from National Center for Medical Culture Collection (CMCC). Bacterial lysis buffer (BugBuster Protein Extraction Reagent) was purchased from Novagen. Lysozyme was purchased from TIANGEN. The wafer was purchased from Avago Technologies. 1,2-Propanediol monomethyl ether acetate (PGMEA) was purchased from Agilent. All other reagents were purchased from Sigma Company. Microfluidic Chip Fabrication. The whole system was composed of two parts: the airborne bacteria enrichment microfluidic chip and the microfluidic immunoassay chip. The airborne bacteria enrichment microfluidic chip was fabricated by multilayer soft lithography processes.13 It included the fluid layer and the herringbone layer which are made of PDMS. These two pieces of PDMS were assembled together precisely.

two technologies and offered a new way for rapid detection of Mycobacterium tuberculosis.20−25 Our lab previously reported a microfluidic system that performs fast and efficient airborne bacteria capture and enrichment.26 The whole system is perfect for field application of a hospital or a high-risk environment with airborne microorganisms. The microfluidic chip is simple and easy to fabricate from plastics by molding. This microfluidic system was demonstrated to have very high-efficiency in bacteria capture and enrichment. The capture efficiency was almost 100% with the function of SHM (staggered herringbone mixer) structure.27−36 Herein we combined enrichment and immunoassay to develop an automatic microfluidic system for airborne Mycobacterium tuberculosis on-chip capture, enrichment, and direct bacteriological diagnosis. To the best of our knowledge, this is the first report of integrated on-chip airborne bacteria capture and microfluidic immunoassay. Compared with the previously reported method of on-chip capture and off-chip analysis, there is no need to wash out the bacteria from the capture chip to perform analysis. The bacteria can be analyzed by the sample-in-result-out manner. In addition, the out chip immunoassay provides better detection efficiency and sensitivity compared with conventional off-chip immunoassay. The combined system also provides advantages such as automation and less human intervention to reduce the experimental errors. The whole system has the potential to become a crucial platform in biological and medical applications for effective and rapid diagnosis of infectious diseases and accurate screening of pathogenic microorganisms in the aerosol. 5816

dx.doi.org/10.1021/ac500578h | Anal. Chem. 2014, 86, 5815−5821

Analytical Chemistry

Article

Figure 2. (A) Schematic illustration of immunoassay reaction for Ag85B. (B) Illustration of the chip operations to complete the immunoassay. The status of the valves was clarified by different colors: red (regular valve) and black (sieve valve) for action; gray for inaction.

of an airborne bacteria enrichment microfluidic chip (Figure 1A,a) and a microfluidic immunoassay chip (Figure 1A,b). The enrichment microfluidic chip were made of two layers of PDMS, containing microchannels with the staggered herringbone mixer (SHM) structure (length 17.4 cm, width 600 μm, depth 40 μm).26 This microfluidic chip had been proved to have an ability of fast capture (within 9 min) and enrichment of airborne bacteria with high efficiency (close to 100%).26 The photograph of the microfluidic immunoassay chip is shown in Figure 1C. It was made of two layers of PDMS containing a fluidic layer (Figure 1D,i) and control layer (Figure 1D,ii). The microchannels of control layer were composed by five individual immune-reaction columns (Figure 1E). One outlet was shared by these columns through a circulation microchannel while each column had its own inlets and microvalves (Figure 1E). These columns could be used independently by controlling various on-chip valves and five different samples could be analyzed at the same time; the operations of the columns could be performed in sequential or parallel manner due to requirements of the experiment. Immune-reactions of different bacteria samples were ensured to work in parallel and independent way on this microfluidic chip. Samples and concentrations interference could be reduced as well. Figure 1F showed the detailed structure of one reaction column. The microchannels of fluidic layer were 25 μm height, 200 μm width. For each column, five inlets were designed for different reagents. The regular valves were used to control the fluid, and the sieve valve was used to intercept microspheres.37 Because of the nanoliter scale reaction volume, the consumption of samples and reagents were reduced compared with the traditional immune-reactions. Mechanism of Airborne Bacteria Direct Capture and Analysis System. Air containing the bacteria was drawn into

More detailed description of the enrichment microfluidic chip fabrication is presented in ref 26. The operation protocol was modified to perform the current analysis. The microfluidic immunoassay chip was fabricated by multilayer soft lithography processes.37 It included the fluidic layer and the pneumatic control layer (Figure 1 D,E). The fluidic microchannels were made of PDMS (RTV 615, the ratio of A/B is 5:1). The pneumatic control microchannels were made of PDMS (RTV 615, the ratio of A/B is 20:1).25 These two layers of PDMS were aligned together accurately and bonded on a glass slide coated with a thin film of PDMS (RTV 615, the ratio of A/B is 20:1). The control layer was both made of negative photoresist (SU8-2025). The mold of the fluidic layer was made from a positive photoresist (AZ-50XT) for the reaction column as well as a negative photoresist (SU8-025) for other channels. Immunoassay Reaction System for Ag85B Detection. Figure 2A shows the schematic illustration of the immunoassay reaction for Ag85B on the immunoassay microfluidic chip. Microcolumns were built inside the microfluidic chip using a specially designed “sieve valve”,37 and microspheres coated Protein A were prefilled as the substrate to perform the immunoassay. The surface of the microspheres was first immobilized with rabbit polyclonal antibody by covalent cross-linking.37,38 In the operation procedure, antigen Ag85B from lysed Mycobacterium tuberculosis, mouse monoclonal antibody, and FITC labeled antimouse secondary antibody were sequentially introduced to on-chip microcolumns to perform the immunoreaction process.

■

RESULTS AND DISCUSSION Design of Airborne Bacteria Direct Capture and Analysis System. The system of airborne bacteria capture, enrichment, and detection are shown in Figure 1A, composing 5817

dx.doi.org/10.1021/ac500578h | Anal. Chem. 2014, 86, 5815−5821

Analytical Chemistry

Article

fluorescence microscope equipped with CCD. Figure 3A showed the image of a homogeneous reaction column under

the enrichment microfluidic chip under the vacuum of a micropump.26 The herringbone shape could induce chaotic flow which created more chances for bacteria capture. Bacteria were adhered to the inside wall of channels efficiently.26,39 As shown in Figure 1B, 2 μL of bacterial lysis buffer was injected into the chip to washing and lysing the bacteria instead of the washing buffer in the previous report.26 Bacteria lysates that collected from the outlet were used for immunoassay. The detection was based on a fluorescence immune adsorption reaction on the microfluidic chip, and the operation procedure of the immunoassay was briefed as following (Figure 2B). For each column, 1 μL of treated microspheres (according to the volume of the column) was preloaded from the inlet with a flow rate of 1 μL min−1. With the intercept of the sieve valve, microspheres were loaded into the channel (green color) to establish the immune reaction column. The inlets were changed from one to another with the control of valves to establish different microcolumns. Treated bacteria lysates were injected into the column to react with rabbit polyclonal antibody on the surface of the microspheres. PBST and 5% BSA were injected for washing and blocking. Monoclonal antibody to Ag85B solution was injected into the reaction column. Washing and blocking steps were repeated. FITC Anti-Mouse secondary antibody solution was then introduced into the reaction column. The microcolumns were washed with PBST for about 5 min. Finally, the fluorescence image of microspheres column was collected under inverted fluorescence microscopy (Nikon Eclipse Ti, Japan) with blue light excitation. The experiment results were analyzed by the software NIS Element. Validation of Microfluidic Immunoassay. The key component for the assay is the microcolumn filled with tiny microspheres. The antigens (Ag85B) were adsorbed onto the surface of the filled microspheres when the lysis solution passed through the columns and antigens got enriched inside the columns. The microcolumn has much smaller dimensions as 25 μm height and 200 μm width, compared with the traditional reactor in centimeter dimensions. The microcolumn provides a much shorter diffusion path compared to traditional reactors even without consideration of the microspheres filled inside the column. The majority volume of the microcolumn is possessed by the microspheres with a 9 μm diameter, and the reaction volume for the reaction is approximately 15 nL for each column excluding the volume of the microspheres. The microspheres are packed tightly (each individual on-chip column contained about 34 000 microspheres) inside the column to further decrease the diffusion path for the reactants. The microspheres also provide a much larger surface area, as approximately 16.67 mm2 for each column compared with the 2 mm2 reactor with same size. Many more antibodies could be immobilized onto the surface of the microspheres to capture the detection targets to improve the sensitivity (about 8 times more antibodies could be immobilized onto the microspheres compared with the blank reaction channel, presumed at the same antibody immobilization ratio). For the analysis efficiency, the immunoreaction rate is related to the diffusion path of the antibodies/ antigens in solution, and a shorter path could improve the reaction rate. The microcolumn structure could eventually improve both the analysis efficiency and sensitivity.25,37 The feasibility of designed microfluidic immunoassay was validated by using Ag85B protein as antigen. A volume of 1 μL of Ag85B suspension with an original concentration of 1 μg μL−1 was injected into the immunoassay microfluidic chip. After immunoreaction, the results were collected by a

Figure 3. (A) Image of reaction column filled with microspheres under visible light. (B) Reaction column excited by blue light using Ag85B as antigen. (C) Image of reaction column using ultrapure water as a control.

visible light. Figure 3B,C shows the fluorescence images of the reaction column when using Ag85B (with a concentration of 1 μg μL−1) and ultrapure water as samples, respectively. It is clear that the immunoassay system showed good performance. When antigen protein Ag85B existed, under the excited of blue light, the reaction column emitted bright green fluorescence under this condition. When using ultrapure water as a control, no fluorescence was detected. This negative result indicated that the interference of nonspecific adsorption of this immunoassay system could be neglected. It was obviously that this microfluidic immunoassay was feasible in rapid detection of Mycobacterium tuberculosis bacteria. Mycobacterium tuberculosis Cell Lysis and Immunoassay. Ag85B protein was known as a secretory protein of Mycobacterium tuberculosis and bacteria should be lysed to release the more Ag85B protein compared with direct bacteria analysis, no mention that the airborne pathogens are possibly in the resting stage with little secretion. In order to obtain a better immunoreaction result, four common used bacteria lysis methods were tested: by BugBuster protein extraction reagent, by lysozyme, by boiled method, and by repeated freeze−thaw method. Reaction conditions and reaction results of these four lysis methods are shown in Table 1 and Figure 4 respectively. Table 1. Conditions of Different Lysis Methods methods

lysis temperature

BugBuster lysozyme boiled repeated freeze−thaw

room temperature room temperature 100 °C −20 and 37 °C

Reaction time was set at 30 min. The result of using lysozyme solution was negative. The reason could be that some additive composition in lysozyme solution destroyed the protein structure, so none Ag85B was detected. Although the other three methods had positive results, the reaction temperature of boiled method was too high to applied on microfluidic chip, repeated freeze−thaw method was complicated to operate and needed cooling apparatus, which was not suitable for portable device. The lysis temperature of BugBuster protein extraction reagent was set at room temperature, so extra heating or cooling equipment was not needed. The operation was very convenient and this reaction temperature made the reaction 5818

dx.doi.org/10.1021/ac500578h | Anal. Chem. 2014, 86, 5815−5821

Analytical Chemistry

Article

the fluorescence images and optical density of the designed immunoreaction system that different numbers of lysed bacteria were utilized as samples as well as the blank control, the positive control, and the E. coli control. It was obviously from the fluorescence images that the blank control and the E. coli control showed negative results, and the BCG samples showed positive results. The fluorescence intensity increased as the numbers of BCG bacteria increasing from 102 to 105 cells. The concentrations of Ag85B proteins in Mycobacterium tuberculosis bacteria cells were far lower than 1 μg μL−1 (positive control), so the fluorescence intensity of samples was not as strong as the positive control. Under the experiment condition, about 102 Mycobacterium tuberculosis cells were confirmed as a positive result, which means that low concentrations of Mycobacterium tuberculosis could be detected directly by microfluidic based immunoassay without culturing step. Moreover, this detection was not dependent on the concentration of Ag 85B, and it relied only on the Ag 85B content in the whole lysis buffer, because of the enrichment of Ag85B by the column design. It provided a possibility to detect Mycobacterium tuberculosis bacteria samples in low concentrations which was almost impossible for traditional ELISA method (about 103−105 cells which need for a positive result). The performance of rapid detection of Mycobacterium tuberculosis bacteria on the immunoreaction microfluidic chip was evaluated. The optical density image showed the relationship between bacteria numbers and optical density. The average fluorescence intensity of each image was obtained by integration. Results showed that the optical density increased nonlinearly with the increasing of BCG bacteria numbers. No good linear relationship was obtained, possibly due to the fact that Ag85B in each BCG cell was not quite the same.40 All the results were based on five parallel experiments and repeated for five times. The error bar for each plot is not quite large. Generally, this was a quite good method for the rapid detection of Mycobacterium tuberculosis bacteria qualitatively and semiquantitatively. Result of E. coli was negative, which showed the good specificity of Mycobacterium tuberculosis immunoreaction system. Direct analysis of Airborne Mycobacterium tuberculosis samples. Aerosols are prepared from suspensions of 106 cell mL−1, 105 cell mL−1, 104 cell mL−1, and 103 cell mL−1 of Mycobacterium tuberculosis and E. coli (control), respectively. After 2 min of aerosol generation, airborne Mycobacterium tuberculosis were captured and enriched by the enrichment microfluidic chip for 20 min. A volume of 2 μL of bacteria lysis buffer was injected into the microchannel for washing and lysing the bacteria and flushing the bacteria lysates into the immunoassay microfluidic chip. It is clear from Figure 6 that under the experimental conditions, Mycobacterium tuberculosis aerosol samples at different concentrations showed positive results and E. coli aerosol samples showed negative results (ODMycobacteriumtuberculosis/ODE.coli > 2.1). The whole system has the capacity to analyze low concentrations of airborne Mycobacterium tuberculosis directly. With the help of the enrichment microfluidic chip, this system could collect enough bacteria at a low aerosol concentration to perform the immunoassay directly. The microfluidic immunoassay chip could perform parallel multiple samples detection and analysis. Compared with the conventional analysis method, the whole analysis time is about 50 min including bacteria capture and enrichment, bacteria lysis, immunoreaction, and results read out, suggesting that this integrated system is suitable for rapid

Figure 4. Fluorescence images of different lysis methods. (A) Treated with BugBuster protein extraction reagent. (B) Treated with lysozyme. (C) Boiled method. (D) Repeated freeze−thaw.

process simple, which was important to microfluidic system. Overall consideration, BugBuster had the best performance in bacteria lysis and in immunoreaction on microfluidic chips. So BugBuster protein extraction reagent was selected as the bacteria lysis buffer. Different concentrations of BCG bacteria solutions were analyzed on immunoassay microfluidic chips. BCG bacteria concentrations were adjusted to 108, 5 × 107, 107, 5 × 106, 106, 5 × 105, and 105 cells mL−1, respectively. 1 μL of these BCG bacteria solutions were taken as samples which contained about 105, 5 × 104, 104, 5 × 103, 103, 5 × 102, and 102 cells, respectively. Meanwhile, 1 μL of E. coli solution containing 5 × 103 cells was set as a control, 1 μL of solution without any bacteria was set as a blank control, and 1 μL of Ag85B solution (1 μg μL−1) was taken as a positive control. Figure 5 showed

Figure 5. Fluorescence images and optical density of the immunocolumn with loading different volumes of bacteria solution. 5819

dx.doi.org/10.1021/ac500578h | Anal. Chem. 2014, 86, 5815−5821

Analytical Chemistry

Article

Laboratory of Atmospheric Particle Pollution and Prevention (LAP3) (Grant FDLAP13006), as well as AEMPC (Grant KHK1202).

■

(1) Lazcka, O.; Del Campo, F. J.; Munoz, F. X. Biosens. Bioelectron. 2007, 22, 1205−1217. (2) Deisingh, A. K.; Thompson, M. J. Appl. Microbiol. 2004, 96, 419− 429. (3) Sasakura, Y.; Kanda, K.; Fukuzono, S. Anal. Chim. Acta 2006, 564, 53−58. (4) Eteshola, E.; Leckband, D. Sens. Actuators, B: Chem. 2001, 72, 129−133. (5) Eteshola, E.; Balberg, M. Biomed. Microdevices 2004, 6, 7−9. (6) Kuo, J. N.; Chiou, C. H.; Lee, W. W. J. Chin. Inst. Eng. 2012, 35, 589−594. (7) Henley, W. H.; Dennis, P. J.; Ramsey, J. M. Anal. Chem. 2012, 84, 1776−1780. (8) Wang, C. H.; Lee, G. B. Biosens. Bioelectron. 2005, 21, 419−425. (9) Yu, L.; Li, C. M.; Zhou, Q. Front. Biosci. 2005, 10, 2848−2855. (10) Murakami, Y.; Endo, T.; Yamamura, S.; Nagatani, N.; Takamura, Y.; Tamiya, E. Anal. Biochem. 2004, 334, 111−116. (11) Baraniecka, A.; Gorska, M.; Kazimierczak, B.; Szmigiel, D.; Szczypinski, R.; Ilnicki, F.; Kruk, J.; Torbicz, W.; Pijanowska, D. G.; Grabiec, P. Prz. Elektrotechniczn. 2010, 86, 5−8. (12) Li, P.; Abolmaaty, A.; D’Amore, C.; Demming, S.; Anagnostopoulos, C.; Faghri, M. Microfluid. Nanofluid. 2009, 7, 593−598. (13) Henares, T. G.; Funano, S.; Terabe, S.; Mizutani, F.; Sekizawa, R.; Hisamoto, H. Anal. Chim. Acta 2007, 589, 173−179. (14) Herrmann, M.; Roy, E.; Veres, T.; Tabrizian, M. Lab Chip 2007, 7, 1546−1552. (15) Yang, K. S.; Kim, H. J.; Ahn, J. K.; Kim, D. H. Curr. Appl. Phys. 2009, 9, E60−E65. (16) Yu, L.; Li, C. M.; Liu, Y. S.; Gao, J.; Wang, W.; Gan, Y. Lab Chip 2009, 9, 1243−1247. (17) Regehr, K. J.; Domenech, M.; Koepsel, J. T.; Carver, K. C.; Ellison-Zelski, S. J.; Murphy, W. L.; Schuler, L. A.; Alarid, E. T.; Beebe, D. J. Lab Chip 2009, 9, 2132−2139. (18) Kartalov, E. P.; Lin, D. H.; Lee, D. T.; Anderson, W. F.; Taylor, C. R.; Scherer, A. Electrophoresis 2008, 29, 5010−5016. (19) Lowe, A. M.; Ozer, B. H.; Wiepz, G. J.; Bertics, P. J.; Abbott, N. L. Lab Chip 2008, 8, 1357−1364. (20) Heyries, K. A.; Loughran, M. G.; Hoffmann, D.; Homsy, A.; Blum, L. J.; Marquette, C. A. Biosens. Bioelectron. 2008, 23, 1812−1818. (21) Ukita, Y.; Asano, T.; Fujiwara, K.; Matsui, K.; Takeo, M.; Negoro, S.; Kanie, T.; Katayama, M.; Utsumi, Y. Sens. Actuators, A: Phys. 2008, 145, 449−455. (22) Sung, W. C.; Chang, C. C.; Makamba, H.; Chen, S. H. Anal. Chem. 2008, 80, 1529−1535. (23) Luo, Y. Q.; Yu, F.; Zare, R. N. Lab Chip 2008, 8, 694−700. (24) Zhao, W.; Zhang, L.; Jing, W.; Liu, S.; Tachibana, H.; Cheng, X.; Sui, G. Biomicrofluidics 2013, 7, 011101. (25) Zhang, J. L.; Liu, S. X.; Yang, P. Y.; Sui, G. D. Lab Chip 2011, 11, 3516−3522. (26) Jing, W.; Zhao, W.; Liu, S.; Li, L.; Tsai, C.-T.; Fan, X.; Wu, W.; Li, J.; Yang, X.; Sui, G. Anal. Chem. 2013, 85, 5255−5262. (27) Yager, P.; Edwards, T.; Fu, E.; Helton, K.; Nelson, K.; Tam, M. R.; Weigl, B. H. Nature 2006, 442, 412−418. (28) Stachowiak, J. C.; Shugard, E. E.; Mosier, B. P.; Renzi, R. F.; Caton, P. F.; Ferko, S. M.; de Vreugde, J. L. V.; Yee, D. D.; Haroldsen, B. L.; VanderNoot, V. A. Anal. Chem. 2007, 79, 5763−5770. (29) Li, Y. B.; Su, X. L. J. Rapid Methods Autom. Microbiol. 2006, 14, 96−109. (30) Phillips, K. S.; Cheng, Q. Anal. Chem. 2005, 77, 327−334. (31) Xia, N.; Hunt, T. P.; Mayers, B. T.; Alsberg, E.; Whitesides, G. M.; Westervelt, R. M.; Ingber, D. E. Biomed. Microdevices 2006, 8, 299−308.

Figure 6. Optical density of airborne Mycobacterium tuberculosis and E. coli samples.

detection of airborne bacteria. Above all, this direct analysis system has advantages including simple operation, short test time, and accurate and reliable analysis, showing the potential applications in rapid detection of airborne infectious disease.

■

CONCLUSIONS A microfluidic bacteriological diagnosis system that integrated airborne bacteria enrichment and immunoassay function was successfully designed and fabricated in this study. Experiment results confirmed that the rapid bacteriological diagnosis system worked well together for bacteria enrichment and sequential immune analysis. The feasibility of the microfluidic system was validated by directly using Ag85B protein as the antigen as well as direct analysis of bioaerosol containing Mycobacterium tuberculosis bacteria. The microfluidic system demonstrated good sensitivity and specificity at various experimental conditions. Besides, this microfluidic device was very convenient to operate; with less reagent consumption (only 1 or 2 μL of each reagent was needed). In addition, the whole detection time was about 50 min including 20 min of enrichment time and about 30 min of immunoreaction time, which was much less than the conventional method which takes about 29 days including the cultivation process and immunoassay on the ELISA plate. This microfluidic device could be applied not only for the detection of airborne Mycobacterium tuberculosis bacteria but also for other airborne pathogens. This method provided an efficient way for direct airborne pathogens rapid detection on a microfluidic chip, and it also provided a possibility for early warning of the spreading of airborne infectious diseases. Meanwhile, the microfluidic chips also offered a possibility for automation or integration with the current analysis system.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ∥

REFERENCES

W.J. and X.J. contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank for the funding support from NSFC (Grants 21377026, 20975025), Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (Grant 708031), Opening Project of Shanghai Key 5820

dx.doi.org/10.1021/ac500578h | Anal. Chem. 2014, 86, 5815−5821

Analytical Chemistry

Article

(32) Yang, L. J.; Banada, P. P.; Chatni, M. R.; Lim, K. S.; Bhunia, A. K.; Ladisch, M.; Bashir, R. Lab Chip 2006, 6, 896−905. (33) Zhu, L.; Zhang, Q.; Feng, H. H.; Ang, S.; Chauc, F. S.; Liu, W. T. Lab Chip 2004, 4, 337−341. (34) Bao, N.; Jagadeesan, B.; Bhunia, A. K.; Yao, Y.; Lu, C. J. Chromatogr., A 2008, 1181, 153−158. (35) Inatomi, K. I.; Izuo, S. I.; Lee, S. S. Lett. Appl. Microbiol. 2006, 43, 296−300. (36) Liu, Y. S.; Walter, T. M.; Chang, W. J.; Lim, K. S.; Yang, L. J.; Lee, S. W.; Aronson, A.; Bashir, R. Lab Chip 2007, 7, 603−610. (37) Lee, C. C.; Sui, G. D.; Elizarov, A.; Shu, C. Y. J.; Shin, Y. S.; Dooley, A. N.; Huang, J.; Daridon, A.; Wyatt, P.; Stout, D.; Kolb, H. C.; Witte, O. N.; Satyamurthy, N.; Heath, J. R.; Phelps, M. E.; Quake, S. R.; Tseng, H. R. Science 2005, 310, 1793−1796. (38) Akerstrom, B.; Bjorck, L. J. Biol. Chem. 1986, 261, 240−247. (39) Sia, S. K.; Whitesides, G. M. Electrophoresis 2003, 24, 3563− 3576. (40) Taniguchi, Y.; Choi, P. J.; Li, G. W.; Chen, H. Y.; Babu, M.; Hearn, J.; Emili, A.; Xie, X. S. Science 2010, 329, 533−538.

5821

dx.doi.org/10.1021/ac500578h | Anal. Chem. 2014, 86, 5815−5821