An aerosol is technically defined as a suspension of fine solid or liquid particles in a gas. 1 Bioaerosols are some kinds of aerosols mainly coming from living organism and containing biological materials such as proteins, bacteria, virus, pollen, biological products or by-products2. Aerosols have many significant and long-time effects on the radiation balance of the earth, the physics and chemistry properties of the atmosphere, the climate and human health. Some bioaerosols are good cloud condensation nucleus and ice nucleus3. Some bioaerosols play important roles in transmitting infectious diseases of human and animals, such as severe acute respiratory syndrome (SARS)4,5. Due to these important effects of bioaerosols, people want to detect them and have worked out many interesting techniques for real-time, in situ monitoring and analyzing bioaerosols in the atmosphere. Fluorescence analysis, PCR and mass spectrometry are common techniques for detecting and characterizing bioaerosols nowadays. In this short term paper, these three techniques will be introduced.
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Fluorescence is the result of photon emission caused by absorption of one or more photon(s) by a molecule or material that goes back to its ground state. Figure 1 clearly shows the processes of excitation and emission in a molecule6. We can see the initial, final and intermediate electronic and vibrational states from Figure 1. The wavelength of the excitation light and the photon emission is varied for different molecules and groups which have different electronic structures. Compared to non-biological aerosols, bioaerosols contain many fluorophores (molecules could generate fluorescence) such as aromatic amino acid residues (tryptophan, tyrosine and phenylalanine which have been discussed in the lecture), flavins7 and reduced nicotinamide adenine dinucleotides (NADH)8.
Figure 1 Jablonski diagram for fluorescence6.
Although the size of bioaerosols is around several microns, the amount of fluorophores in the bioaerosols is still sufficient to generate fluorescence which is intensive enough to be detected. That is the basis of detecting bioaerosols with this technique.
Some of recent fluorescence techniques for detecting and analyzing bioaerosols are developed from the Fluorescence Particle Counter (FPC) which can detect both the fluorescence signal and light scattering of the individual particle9. It works like this: aerosols in the aerosol flow surrounded by a clean air sheath are directed flying individually through a counter in which a laser beam can excite the molecules in these aerosols. The signals reach a beam splitter. The beam splitter will divide the signal into two parts: it reflects the elastic scattering light to one detector (this detector will determine the particle size using elastic scattering light), and provides the fluorescence signal to the other detector (it can measure the intensity and wavelength of the fluorescence). Then, people can know the size and some chemical composition of these bioaerosols. Kaolin particle is a typical kind of naturally formed non-biological aerosol. So, Kaolin particles may appear with bioaerosols. B. subtilis spores are a kind of bioaerosols. From these FPC results, we can see that fluorescence results can distinguish the bioaerosols from typical non-biological aerosols even those naturally formed non-biological aerosols and may appear with biological aerosols at the same time. But this FPC is not able to detect many different kinds of bioaerosols at the same time.
Figure 2 Scheme of Fluorescence Particle Counter9
Figure 3 (left) A histogram of a polydispersion of kaolin particles as measured with the FPC (right) A histogram of B. subtilis spores measured with the FPC. The spores were centrifuged in order to reduce the amount of dissolved proteins, salts, broken cell parts, etc9.
Based on FPC, people developed new techniques, such as aerosol fluorescence spectrum analyzer (AFSA)10 and single particle fluorescence spectrometer (SPFS) for real-time detecting organic carbon and biological aerosols. And single particle fluorescence spectrometer has become a major technique for real-time detecting aerosols now. In the research of Pan, Y. et al11, the UV-laser (263nm) is used to excite the single-particle fluorescence, and fluorescence spectra are measured by the photomultiplier tubes (PMTs) detector. It is an improved version of their previous research SPF. This improved SPF can measure bioaerosols around 1 Âµm, while previous SPF is about 3 Âµm. And the detector can measure the fluorescence as fast as 90000/s. They measured the spectra of 1611835 aerosols and 79.8% of them are smaller than 3 Âµm. Figure 4 shows 1000 spectra of them which belong to different clusters. Many spectra overlap. The sharp signal at 263 nm is caused by the elastically scattered light from individual aerosols and it can reflect the size of aerosols, according to the explanation of the authors. The percentages of particles included in each clusters are noted in each graph.
Figure 4 Normalized fluorescence spectra of single atmospheric aerosols measured Jan 8, 2008 at 10:00 a.m. at Yale University11.
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SPF can continuously monitoring the size and composition of aerosols through measuring the spectra with time and it is relatively inexpensive, so I think it is hopeful and maybe more widely used in monitoring bioaerosols in future. One limitation is that from the spectrum, people can only know about whether sample aerosols contain some types of bioorganic compositions. But people still cannot exactly know the compounds and the amount of each compound in sample aerosols from their fluorescence spectra.
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR) is another biological analytical method focusing on DNA. PCR is used to amplify a certain range of DNA chain which contains a series of nucleotides people want. In the PCR reaction12, the DNA of interest, deoxynucleotide-triphosphates (dNTPs) (A, T, G and C), oligonucleotide primers, Taq polymerase, buffer solution and some other reagents for controlling the conditions of reaction are included. In the whole process, there are many cycles and each cycle mainly has three steps. Step one is denaturing the DNA by heating to 95oC and the double-stranded DNA will become two single-stranded chains. Step two is the annealing step at around 55 to 65 oC. The oligonucleotide primers which can only complement to certain sequence of DNA will bind to the single chain DNA at the region people want. The third step is the extension step. The Taq polymerase will add dNTP to the primer one by one according to the sequence of the template single chain DNA. Thus, new double-stranded DNA appears and it is the accurate copy of a certain region of the original DNA. If this cycle is repeated around 25 times, the amount of the region the original DNA people want will be amplified greatly. So, it is a hopeful way to detecting low amount or concentration of DNA containing sample.
Nowadays, real-time PCR is applied more widely than traditional PCR. Compared to traditional PCR, post-PCR analysis, such as gel-electrophoresis, is not necessary in real-time PCR. In real-time PCR13, the amplified DNA molecules are measured in real time by detecting the fluorescence at the end of each cycle. The fluorescence is generated by the reporter molecules. Reporter molecules are actually dye molecules which bind to the double-stranded DNA molecules. So, the intensity of the fluorescence can describe the relative amount of amplified DNA products.
One example of the application of real time PCR for detecting bioaerosol is the research of Fallschissel, K. et al14. They applied real-time quantitative polymerase chain reaction assay for detecting and measuring the concentration of the airborne Salmonella cells in the livestock housings. Cells were washed with buffer solution and then parafirmaldehyde fixation followed. Then, the solution of cell was mixed with 4', 6-Diamidino-2-phenylindole (DAPI, it helps sample cell emit fluorescence). The sample cells in the solution were filtered onto the polycarbonate filters with vacuum. They used epifluorescence microscope to measure fluorescence emitted by cells and determine the amount of total cells quantitatively. Then, DNA of interest was extracted from sample cells. In this research, PCR amplified the region of the invA gene which is specific for Salmonella. To compare the results, they also used a cultivation approach to measure the concentration of Salmonella cells. Figure 5 shows the frequency of exposure to Salmonella and mean airborne Salmonella concentration from different working areas in a duck feeding industry. We can see the concentrations of Salmonella are quite different among different areas. The concentrations in the office work, stable work and outdoor are out of the detection range. It is reasonable and in our expectation.
Figure 5 Frequence of exposure to Salmonella (%) and mean airborne Salmonella concentration (cells m-3) subject to the working areas in a duck feeding industry. Concentrations were determined by real-time qPCR using the primer system described by Rahn et al. (1992)14
Another example is the research of An, H. R. et al15. They used three measurement methods to get standard curves and applied real-time PCR to measuring the concentration of the organism Escherichia coli Catellani in the air sample. First, they used these methods to determine the total bacterial number in the sample. Then, they did PCR to amplify the DNA of coil E.. After amplification, the coil E was aerosolized and got into water solution. This solution can be diluted by different times and standard solutions of different concentration are prepared in this way. Standard curves were built by plotting each cycle threshold (CT) value against the log of corresponding E. coli cell concentration. Three standard curves are based on three different approaches for measuring cell concentration, culture-based method (CFU), direct light microscopy and epifluorescence microscopy. The cell concentration was determined by three approaches which were shown in Figure 6. And each approach measured both of the cells collected from real air samples and the cultured cells for comparison. "Isolated-Diluted" and "Diluted-Isolated" are two different methods of genomic DNA extraction. We can see that the concentration got from the standard curve based on CFUs is quite lower than results from Epifluorescence microscopy and direct light microscopy. And results of sample from "Isolated-Diluted" and "Diluted-Isolated" DNA extraction are also different. Based on these results, standard curves using culture-based method (CFU) is not very reliable compared to other two methods. Maybe cultured bacteria used in CFU method cannot stand for all bioaerosols in real samples, so the results are significantly lower than other two methods. And the sample preparation (DNA extraction) can also influence the measurement results.
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The sensitivity and accuracy of the measurement of PCR are related to some factors, such as the purity of the PCR reagents and the imperfected copy during the extension step. If the DNA template is contaminated, the PCR will amplify the entire original DNA sample, so the certain region of contamination DNA may also be amplified. That is not what people want. Imperfect copy will affect the efficiency and purity of the amplification of useful region of original DNA12.
Figure 6 The quantification of E. coli in air sample using colony counting, epifluorescence microscopy, direct light microscopy, and real-time PCR based detection methods. The data are average of six trials and the error bars represent standard deciation15.
As we know, mass spectrometry is a powerful analytical method. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) are two ionization methods which appear a little later. The ionization energy of these two methods is low and proteins and peptides can bear it. So, these two ionization methods are frequently used in analysis of proteins and peptides with mass spectrometry16. Now, the widely used mass analyzers in the mass spectrometry are Time-of-Flight m/z mass analyzer and quadrupole ion traps. The main principle of Time-of-Flight m/z analyzers is that all ions get same kinetic energy during acceleration in the electric field, but final velocity is different due to the difference of mass among individual ions. Thus, the time they spend on flying through the same distance towards the detector will be different. In this way, the mass analyzer can distinguish ions with different mass. The principle of quadrupole ion traps is also based on the behaviour of ions in the electric field. Radio frequency (RF) potential is applied to two pairs of opposing electrodes in the ion trap. Ions of all m/z values are stored in this three dimensional quadrupole field. By controlling the RF, ions of all m/z values are filtered out except ions of interest. So when people scan the RF, people can know whether the ions of interest (with a certain m/z value) exist.
Detecting bioaerosols with mass spectrometry is based on detecting biomolecules contained in bioaerosols. So these two methods could also be applied in detecting bioaerosols with some additional steps.
Kleefsman, I. et al. described17 the techniques of single particle mass spectrometry for analyzing bioaerosols. The air sample containing bioaerosols is pumped into the instrument. First, the detection and size determination the sample flow are achieved when the sample flow passes through two laser beams which are from splitting a continuous wave helium-neon laser beam. The particles in the sample are accelerated and the transit time of each particle between they pass through the two laser beam is recorded. That is for the size determination because the transit time (or the velocity) is determined by the size of the particles. Then, they pass through a 266 nm laser light. The amino acid residue tryptophan will emit intensive fluorescence signal under the excitation of this wavelength of laser. Then, these particles fly through a 308 nm Excimer laser for ionization. Positive ions generated in this method are accelerated and fly towards a multichannel plate (MCP) detector which can record the fly time of these ions. Then, all these data will be processed. So, this instrument can show the size, fluorescence characterization and mass spectrum of the bioaerosols in the sample air flow. The schematic diagram of the bioaerosol mass spectrometer is followed.
Figure 8 is the summation of the mass spectra of 50 bioaerosols containing protein cytochrome C (12360 Da) pre-mixed with the matrix sinapinic acid. We can see that oligomers up to 7 molecules could be detected. Although the resolution is not very good, it still could be used for qualitative identification.
Figure 7 The schematic diagram of the bioaerosol mass spectrometer17
Figure 8 MALDI mass spectra (summation of 50 particles) generated from aerosols containing cytochrome C premixed with the mateix sinapinic acid.17
The mass spectrum sometimes can tell which particular type the particle belongs to, but it can not still tell the accurate chemical composition of the particles. The method of ionization, different instruments and other experiment conditions will also affect the results of mass spectrum. The mass spectrum can be very complicated. Sometimes, people use separating approach to analyze it. People summarize the mass spectrum of particles which fall in the same range of fluorescence and then compare the spectra of a certain range of fluorescence from the sample to the spectra of types of particles we have already known. It is not very likely that the spectrum of the sample is exactly same with the spectra we already know. But we still can know some quality information from the spectra.
One of the examples is the research of Mcjimpsey, E. L. et al18. They used the method above. They measured a huge amount of ambient particles at the San Francisco and got the spectra below. After comparison, they found that a small portion of them have fluorescence similar to Nacillus spores and vegetative cells. So, it is likely that there is a small portion of spores and vegetative cells in the sample air. The mass spectrometry method here is kind of sensitive, because the portion of bioaerosols which can emit fluorescence in this sample is relative low.
Figure 9 266 nm fluorescence for particle fully analyzed at SFO (the place of experiments)18
Figure 10 A typical type of spectrum from the SFO using the 266 nm fluorescence excitation a) for region 1 and b) for region 2. c) A typical type of spectrum in all of the 355nm fluorescence regions. d) A more unique spectral type observed in the region 3 of the 355nm. All of these spectra are average of multiple individual particle spectra. e) An average EH (Erwinia herbicola vegetative cells) spectrum and f) an average BG (Bacillus atrophaeus spores) spectrum for comparison.18
In summary, all of these three techniques of detecting and analyzing bioaerosols are based on detecting the biological macromolecules contained in them. Real-time PCR method actually amplifies the certain region of DNA which people are interested in, and then uses the fluorescence method to measure the relative amount of DNA after amplification. From the relative amount of certain DNA, people can know relative amount of some certain types of bioaerosols contained in the air sample. Fluorescence method mainly measures the fluorescence of some fluorophores in biomolecules which are contained in bioaerosols. And fluorescence signal is different among bioaerosols and non-biological aerosols. The combination of fluorescence analysis and mass spectrometry is more and more frequently used in detecting and characterizing bioaerosols. Using fluorescence spectrum, people can quickly know what the general type the bioaerosols sample belong to. Then, mass spectrometry can tell people more detail about the compounds in bioaerosols and maybe people can know which compounds are in the sample. From research above, fluorescence is a good way to quickly identify whether bioaerosols appear in the air sample and what the approximate types of the bioaerosols are. PCR and fluorescence detection can do some relative quantitative analysis, but maybe more accurate methods for creating standard curves for measuring concentration will appear in future.
The sample preparation is another important factor for analysis bioaerosols. The approaches of collecting bioaerosols can affect the total concentration of bioaerosols in the sample. Filter is a common approach. There are mainly two types: fibrous mat type and porous materials and membranes type (such as polycarbonate filters in the research above). Impactor, electrostatic precipitator and sedimentation collector are other methods to collect aerosols.19 They are useful to different types of aerosols and different analysis requirements. Then, preparation of sample in different analysis methods and different calibration may also affect the analysis results.