Bioanalysis within Microfluidics: A Review - ACS Symposium Series

Chapter 13

Bioanalysis within Microfluidics: A Review Downloaded by CENTRAL MICHIGAN UNIV on December 30, 2015 | http://pubs.acs.org Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.ch013

Wenwen Jing1,3 and Guodong Sui*,1,2 1Shanghai

Key Laboratory of Atmospheric Particle Pollution and Prevention (LAP3), Department of Environmental Science and Engineering, Fudan University, 220 Handan Road, Shanghai, 200433, People’s Republic of China 2Institute of Biomedical Science, Fudan University, Shanghai, 200032, People’s Republic of China 3Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, People’s Republic of China *E-mail: [email protected].

Rapid analysis of bioaerosol, including airborne pathogens plays a critical role in bioaerosol study as well as in the early warning of infectious diseases. Related research is essential not only to the scientific society, but also for public health and disease prevention. We present herein the current situation of bioaerosol detection, enumerate the main existing problems, put forward countermeasures and suggestions, and report a series of integrated microfluidic chips and systems that can execute airborne microbe capture, enrichment and continuous-flow high-throughput bioanalysis. Six microbes have been used to validate the capture and analysis efficiency of the system. Our experimental results showed that capture efficiency and detection limit had been greatly improved by microfluidics compared to traditional methods. The capture efficiency of microfluidic chip reaches almost 100%, and the detection limits down to approximately 118 cells were achieved toward Escherichia coli (E. coli), without the DNA purification process. It can collect enough bacteria from low concentration bioaerosol (as low as 100 cfu/m3) for the downstream direct protein/nucleic acid analysis. The whole operation is simple

© 2015 American Chemical Society In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Downloaded by CENTRAL MICHIGAN UNIV on December 30, 2015 | http://pubs.acs.org Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.ch013

and feasible, suitable for on-site application, e.g. at airports, subway stations and hospitals, showing potential application in environmental monitoring and public health protection.

Airborne pathogens are generally defined as pathogens that exist independently or attached to liquid or solid particles suspended in a gas (1–3). For the past few decades, human beings faced threats from all kinds of emerging infectious diseases and old infectious diseases (4–10). In addition to the familiar Tuberculosis (caused by Mycobacterium tuberculosis [TB]), Severe Acute Respiratory Syndromes (SARS), and Avian Influenza (avian flu) (caused by viruses H1N1, H5N1 and H7N9), there are many other kinds of infectious diseases that are caused by various airborne pathogens (11). These airborne pathogens are divided into the following types. The type bacteria includes Bacillus anthracis, Yersinia pestis, Vibrio cholera, Bacillus maller, Tularemia coli, etc. The type viruses includeds Yellow fever virus, Venezuelan equine encephalitis virus (VEEV), Variola virus, Semliki forest encephalitis virus, Dengue fever virus, Lassa fever virus, Rift valley fever virus (RVFV), etc. The type rickettsiae includes Typhus group rickettsiae and Q fever rickettsia (QFR), etc. The type chlamydia includes Chlamydia psittaci. The type fungi includes Aspergillus flavus, Aspergillus niger, etc. (12, 13) Infectious diseases are difficult to control and prevent worldwide because their pathogens can spread fast among people through the air as the medium (14). When exposed to air polluted by pathogenic bacteria, the human body could be invaded through the skin or mucous membranes or respiratory tract and become infected (15). Human health is threatened and damaged frequently, not to mention the public panic and economic losses accompanying airborne pathogens, which are gaining increasing interests in recent years. Methodology for rapid or on-line detection of airborne pathogens is essential for disease control and prevention. In this review, we will provide an overview of detection of airborne pathogens, the challenges, and the future perspective.

Challenges in Airborne Pathogen Analysis So far, identification of airborne pathogens still relies on the clinical observation and follows precise molecular and immunological diagnosis of blood samples from potentially infected patients (16, 17). This process normally takes days, which is too slow for prompt disease control and prevention. The rapid and direct analysis of airborne pathogens helps disease control and surveillance resources for pandemic diseases, especially for highly populated metropolitan areas (18, 19). However, there are still technical obstacles even with great development in molecular biology in the past couple decades. The obstacles come from two aspects of airborne pathogen analysis: initial airborne pathogen collection techniques and downstream bioanalytical techniques for identification of collected pathogens. 246 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


The sampling procedure is a key to get a reliable observation in the application of airborne pathogen analysis (20, 21). The selection of sampling equipment is an important issue in this procedure to ensure the collection efficiency is directly related to the accuracy in the evaluation of final analysis results. According to practical requirements of sampling, sensitivity, stability, reliability, portability, and ease of operation, maintenance and analysis are issues that need to be considered. The first airborne pathogen natural sedimentation method was first developed by Koch in 1881, where airborne pathogens were settled by gravity on nutrient agar media (22). It is the simplest and most economical sampling method in which the results roughly reflect the structure and amount of airborne microorganisms. However, the defects of natural sedimentation methods are obvious. The sampling conditions are hard to control and may induce great measuring error because of interferences such as wind force, electric force, magnetic force, thermal force, buoyancy force, and diffusive force. Besides the airborne pathogen natural sedimentation method, there are machine sampling methods for airborne pathogens (23). For example:

Anderson Sampler The Anderson sampler is the widely used airborne pathogen sampler which can not only measure the approximate size of the pathogens, but also their concentration (24). The sampler size ranges from 0.2~20 µm which is able to satisfy most pathogen sampling requirements except for those pathogens that could not be cultured on various media. However, operation is complex and needs a large number of culture plates. According to the different growth time of various pathogens, the culture time ranges from several days to several months before the concentration of pathogens fulfills the detection requirement (25). If pathogens are sensitive to air pressure, it is difficult to keep these microorganisms alive.

All-Glass Impinge (AGI) Sampler The AGI sampler is a glass collection device that is designed to collect airborne pathogens with a high concentration (26). It is operated by drawing airborne pathogens through an inlet tube and subsequently passing through a jet into a liquid medium (27). Take AGI-30 for an example, the jet is positioned 30 millimeters (mm) above the bottom of the glass impinger, and consists of a short piece of capillary tube. The medium of this sampler could protect the pathogens from losing their biological activity (28). All airborne pathogens drawn into the AGI-30 sampler could be contained efficiently (29). This sampler is only suitable for airborne pathogens with a high concentration. When the concentration of pathogens is low or when the temperature is low, it is hard to detect airborne pathogens successfully. 247 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Filter Sampler


The Filter sampler is an air extracting instrument into which airborne pathogens could be intercepted by a filter membrane when bioaerosol passes through the membrane under negative pressure (30). The core component of this equipment is the filter membrane. According to different pore size and materials of the filter membrane, various airborne pathogens could be collected on the membrane with a high collection efficiency (31). However, this instrument is not suitable for airborne pathogens which are intolerant to dry conditions. The efficiency of the sampler may also be influenced by the particle elution process.

Static Sampler Vaporized bacteria carry electric charges, which could be used for bacterial aerosol collection, according to the electrostatic adsorption principle, in the static sampler (32, 33). This equipment contains a high voltage power supply, discharge electrodes, collecting electrodes and air extracting device. The sampling progress of this instrument has advantages such as retained bioactivity of aerosol bacteria due to its big sampling volume and high concentration ratio. However, ultraviolet light, ozone and nitrogen oxides produced during the sampling process will degrade the microorganisms (34). The big and complex instrument has some disadvantages such as inconvenience of installation and maintenance, and carries safety risks, which detract from practical application. Although many different types of samplers are promoted for the development of air microbiology, so far, fast and accurate sampling of the biological aerosol is still a difficult technical issue (35, 36). Almost no sampling technique could ensure that the collected microbial specimen reflects its original state and can be directly used in bioanalysis (37). Pathogen culturing is generally the necessary step before analysis, mainly because the concentration of collected pathogens is too low for direct bioanalysis for the methods mentioned above. Before the inventions in molecular biology, physical and chemical, airborne pathogens were mainly studied through cultivation methods on selective and nonselective media. These methods have been used in the study of aerosol analysis with a long history and will still be used as useful analysis tools in microbial aerosol particles analysis.

Cultivation Methods Cultivation methods can only detect certain viable airborne microorganisms in the air such as some living bacteria, fungi and algae. However, after death, these bacteria, fungi and algae, as well as their fragments, such as cell walls or cytoplasm materials are incapable of being detected or collected by cultivation methods. Furthermore, cultivation methods are only suitable for detecting and collecting those microorganisms that can grow on culture medium (38). However, studies of the cell viability of environmental microorganisms have found that the vast majority of viable microorganisms that exist in ambient environment are in 248 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


viable but non-culturable state (39). Only about 17% of known fungi species can be cultivated on the culture medium (40). When it comes to bacteria, only less than 10% of the total number of bacteria can be cultivated, which can be observed in the range of about 0.01% to 75%, with about 1% as its average values (41). Therefore, studies based on microbial cultivation methods often largely underestimated the microbial diversity and concentration of microorganisms. In addition, when microorganisms are collected by conventional sampling methods, this can cause a great damage on a number of viable cells, because the suction pressure generated in the sampling progress will lead to some viable cells death (42). The culturability of living microorganisms also decreases rapidly through the traditional sampling methods (43). Finally, the number of microorganisms are likely to be greatly underestimated, because one single aerosol particle may represent at least one kind of a microorganism’s clone, whereas the traditional sampling methods and cultivation methods will only form one colony on the culture media (10, 44, 45). Nonetheless, there are limitations, cultivation methods have the advantage that they are cheaper than molecular biology detection methods, and cultivation methods can give an indication of the quantity of viable microorganisms cells in the air, whereas molecular biology methods can only prove the existence of these microorganisms but are unable to distinguish whether these organisms are alive or dead (46–49). Cultivation methods are particularly suitable for detecting certain groups of microorganisms or targeting individual species, and collecting microorganism cells for culture collections (50–53). Culturing normally takes from 24 hours to several days to more than ten days, which is too slow for rapid analysis, especially for the early disease warning and prevention situation (54). Some bacteria were in the state of viable but non-culturable (VBNC) in natural environments (55). Most pathogens are sub micron in size, and the dimensions of the current samplers are at millimeter or centimeter level. Because of the size difference, the water used to rinse/wash the samplers is generally excessive, compared with the small amount of the pathogens collected in the samplers. Consequently, the concentration of the collected pathogens in the aqueous media is too low for direct bioanalysis.

Microscopy Techniques As extremely valuable research tools, microscopy techniques have played very important role in the analysis and detection of microbial aerosol particles (56). Various types of optical microscopy were applied in the classification and characteristics description aspects of the collected microbial aerosol particles (57). However, it has to be taken into consideration that when the particle size is smaller than 2 microns, it only appears as a dot, and its characteristics size, shape, and detailed microscopic structures could not be distinguished and analyzed clearly under optical microscope (58). Therefore, such kind of aerosol particles cannot be classified and analyzed by an optical microscope. The direct number counting and strain identification of microbial aerosol particles are both very tedious processes. It will be influenced by some subjective factors when counting the microbial aerosol particles with naked eye. For the species diversity 249 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


analysis of microorganisms, the credibility of microscopic analyses results is usually not high. In the identification of strains, some of the strains are similar in morphological characteristics, which causes difficulty in species differentiation. Some microorganisms can only be identified as belonging to broad categories or a specific family (59). Fluorescence microscopy is often used to observe microbial aerosol particles which are capable of autofluorescence (60, 61). Microbial aerosol particles also could be observed by fluorescence microscopy after labeling them with fluorescent dye (62, 63). The most commonly used traditional methods for counting the total number of environmental microbes are by direct counting under fluorescence microscopy. This counting is usually achieved by detecting the autofluorescence of certain biological compounds or by detecting the fluorescence of microorganisms that are treated with some commonly used fluorescent dye, such as 4.6-diamidino-2-phenyl (DAPI) and acridine orange (64, 65). Recently, more and more automated analysis techniques such as computer analysis of microscopic images and fluorescence spectroscopy have been applied in microorganisms detection fields combined with fluorescence microscopy (66–70). However, sometimes fluorescence microscopy is unable to clearly distinguish different types of biological particles, such as fungal spores and bacteria (71).

Chemical Tracers Chemical tracers, such as sugar alcohol, mannitol and arabitol are used as chemical tracers for assessing diversity of not only microbial aerosol but also other kinds of aerosol particles (72–74). These chemical tracers can be combined with multiple bio-analytical techniques including liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometer (GC-MS), ultraviolet spectrophotometry, fluorescent spectrophotometry, immunoassay, and dyes analysis for sample testing (75–77). The merit of chemical tracer analysis is quantification of information although biodiversity or identity of microbial aerosol particles at species level is generally not available (78).

Nucleic Acid Sampling and Extraction from Bioaerosols Microbial aerosol particles are gathered and extracted from samples including solid medium, liquid medium, water, biological aerosol particle collectors or air filters before analysis of microbial aerosol with tools of molecular genetics. Successful microbial DNA extraction is the basis of analysis with molecular biology method (79–82). Microbial protein is denatured and mixed with liquid, pigment, fragments of cell wall and organelles in the process of DNA extraction (83). DNA of microorganisms with thick membrane or cell wall is extracted insufficiently with above-mentioned protocol and the quantity of such microorganisms is underestimated in environment samples with diversified biological materials (84–88). 250 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Amplification of Genomic DNA The DNA of the target microorganism is supposed to be gathered so as to make the quantity of it much higher than that of other ones for the recognition of some single genera or species (89). DNA characteristic regions of one or several species of microorganisms are effectively amplified and analyzed with polymerase chain reactions (PCR) in which DNA double strand is denatured by heat and a corresponding single strand in target genome is paired with short DNA sequences called primers according to the base complementation pairing rule and finally a new complementary strand is assembled with mononucleotide in target region by DNA polymerase within one heating cycle (90). The amplified sequences in PCR are useful for research on species identifying, specific genes or phylogenesis (91). As it is, genomic DNA amplification is considered to be important in investigating constitution and diversity of microbial aerosol although not all DNA of every single species in atmosphere could be amplified equally with above-mentioned method (92). DNA polymerase is capable of amplifying sequences from even a single copy of some strand theoretically so as to be highly sensitive in detecting trace quantity of environmental microorganisms although practical sensitivity depends on several factors including specificity of primers and integrity of DNA samples (93, 94). So as to say, one copy of a single-strand target gene is enough to start amplifying with highly specific primers while one hundred times of that is enough with not-so-sensitive primers (39, 95, 96). Specificity is extremely important for precise amplifying and high signal-to-noise ratio. Although primers designed for given DNA sequences are supposed to attach exclusively to the specific region in the target genome, they are still possibly combined with non-target sequences because of several times and even more quantity of competing genes (96). Although DNA molecules are relatively stable in cool, dark and dry condition and are possible to be conserved for even up to thousand years, the degradation process starts as soon as life ends (97–99). DNA molecules are reduced into smaller fragments and chemically modified and the process is accelerated by ultraviolet light, ozone and freezing-thawing. Degradation of DNA and loss of genetic information is caused by the exposure of biological materials to air for a long time (100, 101).

Restriction Fragment Length Polymorphism Techniques (RFLP) Restriction endonuclease is a kind of enzyme which is capable of recognizing and cutting specific sequences of a molecule at special sites producing restrictive fragments (102, 103). This is the theoretical basis of the technique (104–107). Genomes of different species are distinct from each other. Difference between genomes lying in a cleavage site of a restriction endonuclease leads to imparity in recognition of enzyme and causes diverse characteristics of digested fragments in length and quantity (108). Polymorphism of these fragments is analyzed with a whole set of techniques including electrophoresis, trans-membrane, denaturation, hybridization with labeled probe and washing membrane (109–113). This protocol is supposed to be used after colony PCR (make sure the plasmid has been 251 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


inserted into bacterial genome) in order to collect as many colonies as possible for sequencing (114–118). T-RFLP is used to investigate microbial community and estimate diversity and relative abundance of aerosol samples roughly (119, 120). First, the target sequence is attached with fluorescence-labeled primers and amplified in PCR. Then PCR product is digested with restriction enzyme at target site producing shorter labeled fragments. Fragments differ in lengths because of diverse positions of target sites in bacterial genomes from different species. Fluorescence-labeled fragments are separated with electrophoresis and lengths and fluorescence intensity of them are measured. Fluorescence of a DNA strand with a given length reveals the concentration of the corresponding bacterial strain in original samples. Sequencing Methods Product of PCR is usually cloned and sequenced to identify genomes in atmosphere aerosol samples. The sequences are then compared with known sequences from on-line database in order to determine the species of microorganisms in samples (121–124). If a new species is found (for example, a new bacterial strain), genome sequences of its nearest relative could be found from on-line database. Generally accepted interspecies difference in bacteria is similarity of 97-99% and intergeneric difference is similarity of 95-97% (42, 43). Chain termination is used in conventional sequencing techniques. The quenching is similar to PCR steps only in which the number of primer is one rather than that of two in PCR (125–128). Therefore it is almost impossible to identify microorganisms in biological aerosol particles at species level with current high through-put techniques (129, 130). Microarray Technology Microarray technology is applied to investigate features of microorganism aerosol particles (131–133). Specific probe for species or population is fixed on a glass chip (134–137). Fluorescence-labeled DNA in atmosphere samples is supposed to hybridize with complementary DNA sequences on the chip if the sequences exist (138, 139). Gene sequences are determined by the position of fluorescence signal on the chip (140, 141). On-Line Auto Fluorescence Methods This technique is currently applied to the determination of 16S rRNA in bacteria from atmosphere aerosol samples. It is helpful to determination if biological material contains fluorescent substance. Ultraviolet Aerodynamic Particle Sizer (UV-APS) is the first commercial instrument that is capable of analyzing biological aerosol samples on line based on fluorescence (142). The aerodynamic diameter and lateral scattering parameter (similar to optical diameter) of particles is measured by testing flight time (633nm) of particles between two laser beams and by fluorescence with the wave length range of 420-575nm excited by pulsed UV laser (Nd: YAG, 355 nm). Wide Issue 252 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Bioaerosol Spectrometer (WIBS) is also produced in a limited run providing not only similar information with UV-APS but also a rough estimation of the sphere factor (143). The size of incident particles is determined by measuring with a scattering laser at first (144). Fluorescence emission spectrum at 310-400 nm (excitement wave length of 280nm) and 420-650 nm (excitement wave length of 280 and 370nm) is recorded with every particle excited by UV pulse by a xenon flash lamp of 280 and 370 nm (145). Report on environment monitoring with this technique is still rare although development of it has been published in military report and peer review (146). Flow Cytometry Flow cytometry has always been an important tool in research on environmental microorganism aerosol particles (147–151). In reports of testing biological aerosol on line using Flow cytometry successfully, there are many data about research on features of atmosphere microorganism using Fluorescent in situ hybridization (FISH) flow cytometry and about research on features of aerosolized by-product indicating the existence of bacteria spores and fungi (152). Light Detection and Ranging (LIDAR) and Remote Sensing The LIDAR technique, also called microwave radar, is a technology that measures distance by illuminating a target with a laser (including ultraviolet, visible, or near infrared light) and analyzing the reflected light (153–155). It has been utilized to quickly and remotely monitor the presence of microbe aerosol particles over a larger spatial range. A LIDAR system was operated to determine its sensitivity to aerosolized Bacillus subtilis spores (156, 157). Mass Spectrometry (MS) The Mass spectrometry technique has been applied to many areas in physics and biology in the past decades providing detailed chemical composition information (158, 159). Many different MS techniques have been applied to research on characteristics of microbial aerosol particles (20, 160, 161). Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) has been applied to multiple researches on microbial aerosol successfully (162–166). Laser-Induced Breakdown Spectroscopy Laser-induced breakdown spectroscopy (LIBS) is one type of atomic emission spectroscopy which uses a highly energetic laser pulse as the excitation source. Many groups have also utilized LIBS to identify elemental composition as means of biological aerosol detection and analysis, although these have not been as widely applied to ambient measurements (167–170). LIBS technique has been applied to research on characteristics of diverse microbial aerosol particles including pollen, bacterial spores and fungi (167–170). Other form of elemental analysis including spark-induced breakdown spectroscopy (SIBS), particle-induced X-ray emission 253 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

(PIXE) and combustion analysis have been applied to experiment research on biological aerosol (171–175).


Microfluidics for Airborne Pathogen Analysis and Remaining Technical Challenges Microelectronic chip and micro total analysis system were the most profound technology in the 20th century history (176–179). The microelectronic chip began to act as the heart of computer and appliance 40 years ago (180, 181). Micro total analysis systems (µTAS) which is also called Lab on a chip or Microfluidic chip was started in the 1990s and is based on micro electromechanical systems (182, 183). Microfluidics, a new emerging technique dealing with samples in micron size and in nano liter volume, is widely applied in protein analysis and nucleic acid analysis (184, 185). It can provide rapid downstream bioanalytical methods as well as initial airborne pathogen capture methods, which fits perfectly for airborne pathogen analysis (186, 187). There are a variety of reports about the microfluidic pathogens analyzed in aqueous medium, including viruses, bacteria and fungi. Characteristic antigen (protein) immune analyses as well as nucleic acid analysis by hybridization or PCR were both successfully performed in microfluidic chips (188). Recently, the capture and enrichment of the airborne bacteria by microfluidic chip was also reported (189, 190). By converting the laminar flow to twisted air flow inside the microfluidic chip to increase the contact opportunity between the channel wall and the bacteria in the airflow, the microfluidic chip can collect hundreds of bacteria within a couple microliters (μL) of aqueous media, sufficient concentration and amount for direct immunoanalysis or nucleic acid analysis. As a new technology emerging in recent years, microfluidic chips which have become an integral part of science and technology and are undergoing a fast development stage, will continue to play an important role in the process of technological development. As an interdisciplinary and cutting-edge science, microfluidic chip technology is widely involved in different fields and techniques (191). It is involved in methods and technology belonging to chemistry, biology, physics and so on (192, 193). In order to achieve the functionalization, techniques from those disciplines were applied alone or fused to the development of microfluidic chips (194, 195). Microfluidic immunoassays. Immunoassays have been used widely in varieties of applications (196). For example, they have been used in environmental, food safety testing, pharmaceutical analysis, medical diagnostics and fields of basic scientific investigation, because they are very sensitive, simple and specific (197). Antibodies (Abs) are proteins produced in animals and human bodies by immune responses. Antigens (Ags) are some kinds of invasive foreign substances to human and animals bodies (198). In nature, these Ags have a highly specific affinity for their cooresponding antibodies. In lock-and-key mechanism, each Ab has a unique structure recognized by a corresponding Ag. There are a variety of formats of these immunoassays. All formats are making use of the sensitivity and specificity of that Ab-Ag interaction (198). The specific Ab-Ag 254 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


interaction allows for the quantification and monitoring of drugs and metabolites and other small molecules such as large proteins, nucleic acids and even whole pathogens (198). In recent years, a promising platform has been extensively explored for the combination of immunoassays and microfluidics as microfluidic immunoassays (199). Most immunoassays include a series of processes, such as washing, mixing, and incubation steps. All those processes are time consuming and need high labor intensity (200). These processes often take several hours. Sometimes one single assay even needs about two days to perform (201). The reason that immunoassays require a long time is mostly attributed to the incubation time, which is inefficient, as well as mass transport process of immune agents from solution to solution surface where the conjugation occurs, but the immune reaction itself is relatively rapid (202). Besides, the immune agents used in immunoassays are very expensive. If the system is miniaturized, the consumption of the immune agents will be reduced largely. Therefore, microfluidic immunoassays which are automated and miniaturized are in great demand. They have simplified the procedures, reduced the assay time as well as the sample and regent consumption, and at the same time enhanced the reaction efficiency. Recently, extensive investigations using microfluidics in the immunoassay field have been reported (203, 204). Isothermal Nucleic Acid Amplification in Microfluidic Platforms. Isothermal amplification methods use the enzymes involved in the synthesis of DNA/RNA in vitro in a thermostatic reaction. Many isothermal nucleic acid assays have been developed with different types and different numbers of enzymes and primers, amplification times, incubation temperatures and detection methods (205). Isothermal nucleic acid amplification assays such as loop-mediated isothermal amplification (LAMP), multiple displacement amplification (MDA), helicase dependent amplification (HDA), recombinase polymerase amplification and nucleic acid sequence-based amplification (NASbA) have also been translated on microfluidic platforms (206). LAMP is different from PCR. The polymerase extension rate is the limiting factor in the isothermal nucleic acid amplification assays. The detection times of these assays in conventional detection formats are considerably high, ranging from 30 to 90 min (207, 208). The detection time was reduced to less than 30 min by several factors, such as the optimization of primer design and reaction, the application of highly fluorescent dye and the improvement in detector sensitivity. Microfluidic based isothermal nucleic acid amplification assays are simpler and less expensive because they don’t require any sample or temperature circulation. LAMP is first reported in 2000 as a relatively new gene amplification technique. Appealing features of LAMP include high sensitivity, high specificity, speed, and high product yield. LAMP is performed at a moderate incubation temperature between 60 °C and 65°C. Major reaction components are as follows, four primers and an enzyme (Bst polymerase) with strand displacement activity. The addition of two more primers called loop primers further enhances the sensitivity and speed of LAMP reactions. Amplification times of tube-based LAMP assays typically vary between 30 and 90 min, depending on the starting DNA template. Endpoint and real-time LAMP assays employed with turbidity or fluorescence-based detection schemes 255 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


have also been translated on microchips made of silicon and polymer. Positive endpoint microLAMP is confirmed either visually or by fluorescence microscopy. Fluorescence or turbidity increment during real-time microLAMP is commonly monitored by photodiodes. MicroLAMP is further quantified by measuring the threshold time (Tt) for a selected cutoff of signal, where the signal is above the background (209, 210). Although microfluidic techniques solved many of the problems for the conventional airborne pathogen analysis from sampling to the downstream bioanalysis, however, many challenges remain. Pathogens larger in size, such as bacteria and fungi can be easily trapped inside the microfluidic chip; there is still no report about the collection efficiency of the virus from air, possibly because of its nanometer size, which is much smaller than bacteria and fungi. Since many common respiratory infectious diseases, such as all kinds of flu, are caused by virus, there is great technical need for methods capable of high capturing efficiency of virus in microfluidic chips (14, 211). In addition, the proper salt concentration, pH values as well as relative environmental cleanliness are all essential for successful downstream immunoanalysis or nucleic acid analysis. Most airborne pathogens exist within particles and constitute only a tiny part of the particles (2). The major components of the airborne particles are dusts, salts and some organic compounds. The various species and ratios of these components are determined by the individual environments of the particles (211). The aqueous media used in the direct collection of the pathogen from the surrounding air sometimes is not suitable for direct bioanalysis. It is necessary to develop microfluidic methods capable of separating pathogens from particles without greatly changing the total volume. Furthermore, because of the many types of airborne pathogens to screen in the case of a cautionary situation, high throughput downstream analytical techniques within microfluidic chips are in great need (212). However there is no available system for direct accurate airborne pathogen analysis, there are still parts missing to connect the microfluidic sampler with microfluidic chip for bioanalysis. The connection between them as well as the systematic automation will be another challenge to be conquered before any commercial instrument is developed. Herein our group has reported series of integrated microfluidic chips and systems that can execute airborne microbe capture, enrichment and continuous-flow high-throughput bioanalysis. Our group has established a simple, cheap polydimethylsiloxane (PDMS) microfluidic device which is capable of rapid and efficient airborne bacteria capture and enrichment. The device consists of a two layer PDMS microfluidic chip with two plates of polymethyl methacrylate (PMMA) bonded to the upper and lower surfaces of the microfluidic chip. This microfluidic chip consists of an inlet, an outlet, and the inside enrichment channel. The core capture channel is 17.4 cm long, 600 μm wide, and 40μm high. The capture channel is designed as an s-shaped zone and chaotic flow zone. A staggered herringbone mixer (SHM) structure has been used in airborne bacteria enrichment on a microfluidic chip for the first time. The SHM structure is designed to create chaotic flow and increase the contact chances between the bacteria in the aerosol and the inner walls of the microchannels inside the microchip. This device was validated with 256 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


E. coli and Mycobacterium smegmatis. The capture efficiency of the microchip reaches almost 100% in 9 minutes. The capture limit is lower than the plate sedimentation method. This device can collect enough airborne bacteria for a direct test. The whole system is perfect for field application especially in some airborne microorganisms’ high-risk environment. It has the potential to become a crucial platform for aerosol microorganisms detection (213). Based on this work, we developed a rapid capture, enrichment, and direct bacteriological diagnosis method for airborne Mycobacterium tuberculosis bacteria with Enzyme-linked Immuno Sorbent Assay (ELISA) double sandwich method based on microfluidic chip. The microfluidic immunoassay chip was made of two layers of PDMS containing a fluidic layer and control layer. Five immune-reaction columns were designed to analyze five different samples independently at the same time. The operations of the columns could be performed either in sequential or parallel manner according to the experiment requirements. The microchannels were 25 μm high, 200μm wide. The fluid was controlled by regular valves and siece valves. The key component for the assay is the microcolumn filled with tiny microspheres in 9 μm diameter. The reaction volume for each reaction is only approximately 15 nL. This immunoassay system could successfully identify antigen protein Ag85B, and is very convenient to operate, with little reagent consumption (only needs 1 or 2 µL of each reagent). The detection time was about 35 minutes which is far less than the traditional ELISA reaction on 96-well plates. Only 1 µL to 5 µL sample containing about 100 to 500 cells is needed for the detection of positive results. Compared with the previously reported method of on-chip capture and off-chip analysis, their is no need to wash out the bacteria from the enrichment chip to perform analysis. This device could also be applied for the detection of airborne pathogens and provide a possibility for the early warning of the spreading of airborne infectious diseases and for the automation platform with this analysis system (214). We also presented a simple and low cost continuous-flow PCR device by using microfluidics and molecular biology technology. The device is capable of fast detection of environmental pathogens in field. Compared with the conventional PCR method, the required reagent and sample has decreased to 10% by using the presented method. Furthermore, a two-step PCR decreased the processing time by 1/3, and the structure of the chip is simpler. The device is more portable and is easier to operate, which makes it a promising platform for environmental bacteria identification. A high-throughput continuous-flow PCR chip was also developed for airborne bacteria detection. The integrated microfluidic device can perform airborne pathogen capture, enrichment and gene analysis in 2 hours. Six frequently encountered bacteria, including Klebsiella pneumoniae, Citrobacter koseri, Staphyloccocus aureus, E. coli, Pseudomonas aeruginosa, Enterococcus faecalis and Mycobacterium smegmatis, have been used to validate the capture and analysis efficiency of the system, using only 0.13 µL sample for each bacteria analysis. To the best of our knowledge, this is the first report of integrated on-chip airborne bacteria capture and molecular identification. This device can not only be utilized for analysis of multiple samples, but also for multiple analyses of a single sample. The reported device shows great potential towards applications in environmental analysis fields. The whole operation is simple and feasible, suitable 257 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

for on-site application, e.g. at airports, subway stations and hospitals, showing potential application in environmental monitoring and public health protection (215, 216).


Future Perspective It must be mentioned, that microfluidics is not a technique capable of detection of airborne pathogens by itself, and instead, it provides a platform in micron size to facilitate sampling and downstream bioanalysis. Integrating with other new techniques is the key to deal with the challenges. For the efficient downstream characteristic antigen analysis, the protein microarray may hold potential for high throughput immune analysis if the necessary antibodies are available. Rather than protein analysis, new PCR technique, such as LAMP are attractive techniques with high sensitivity and selectivity while requiring less assay time. More importantly, the LAMP assay amplification is carried out under isothermal conditions, and the results can be displayed as ultraviolet-visible (UV-VIS) absorption or fluorescence without the need of conventional hybridization or electrophoresis. The LAMP assay can be easily adapted to a microfluidic chip and various pathogens were already analyzed in the microfluidic LAMP assay (217). Besides the antigen analysis of immune response and detection of target nucleic acid by analysis of pathogens, MS might be another promising technique which can provide rapid response from as little as a drop of medium, perfectly matching the volume of output from a microfluidic chip. Although current MS systems for aerosol analysis are mainly focused on inorganic and organic fragment analysis, the integration of microfluidic sampling and MS will be a very promising platform for airborne pathogen analysis, because of its advantages such as rigid structure, less bio-reagent requirements, temperature control, rapid response and easy automation. Another technical issue for MS is that most MS analysis results can be affected by salt in the buffered medium and it has difficulty in analyzing sample mixtures (218). This problem could be solved by adding microfluidic chromatography after the microfluidic sampler and before loading into the MS ionizer for the airborne pathogen analysis. The most instrumental progress is driven by great need from society or commercial markets. Airborne pathogen analytical systems are directly related to public health and have huge potential applications in disease control, clinical settings, and national security. The airborne pathogen analytical instrument based on microfluidic techniques might make great contribution in this field because of its unique properties compared with traditional techniques. Nowadays, life science, environmental science and medical science are going through the process of development from macro to micro. Biological and chemical equipments were miniaturized and integrated, a series of their functions were transferred from the analysis laboratory to a Lab on a chip. This miniaturization and integration could reduce the dosage of sample and chemical regents, as well as the costs, and energy consumption. Meanwhile it could increase the sensitivity of detection, shorten the test time, and achieve high-throughput. Microfluidic chip 258 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


techniques have integrated with traditional detection methods such as ELISA, PCR and immunofluorescence assay for proanalysis. In the near future, microfluidic chips will be applied to more fields than scientific research, and will also be transformed into products with more subdivisible functions as the development of science and technology. Analytical chemistry would be liberated from the dependence on large equipment in the laboratory and become implemented in common tools used in our daily life in the future. It is believed that microfluidics will become a popular detection-method because of their low cost and ease of application. In the environmental field as well as all kinds of point of care closely related to the safety and quality of our lives, such as water quality, pesticide residues on fruits and vegetables, infectious diseases, pathogenic microorganisms and air quality shall see improvements in the detection of airborne pathogens. We human beings would live a better life with the help of microfluidic chips.

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