Abstract
Disclosed herein are devices and methods for the real-time detection of aeropathogens. The device includes an aerosampler having an air inlet and at least one collector tube, a microfluidic system which includes a container, piping, a micro-pump for flowing a liquid, and a viral detection chamber. The viral detection chamber has an electrode which may be equipped with functionalized biosensors, a counter electrode, an electronic detection system connectable to the electrodes of the viral detection chamber, and an embedded electronic processing system for processing data from the electronic detection system.
Claims
1. A method for real-time detection of aeropathogens by providing an aerosampler having an air inlet and comprising at least one collector tube, a microfluidic system comprising at least one container, piping and at least one pump for flowing a liquid, at least one viral detection chamber, the at least one viral detection chamber having at least one working electrode being equipped with functionalized biosensors and at least one counter electrode, at least one electronic detection system connectable to the electrodes of the at least one viral detection chamber, and an electronic processing system processing data receivable from the at least one electronic detection system, the method comprising: moistening the walls of the at least one collector tube, introducing air into the aerosampler, collecting aeropathogens at the moist walls of the at least one collector tube, flowing moisture from the walls of the aerosampler to the viral detection chamber, detecting electric response between the electrodes of the viral detection chamber, and processing the electric response in the electronic processing system to identify the presence of aeropathogens in the air.
2. The method according to claim 1, wherein two size distributions of aeropathogens particles and/or droplets are collected simultaneously with the aerosampler.
3. The method according to claim 1, wherein the inner walls of the at least one collector tube is continuously wetted with an adequate biofluid for the targeted aeropathogens.
4. The method according to claim 1, wherein the viral detection chamber can be repeatedly cleaned with pure H.sub.2SO.sub.4 acid without any damage or alteration after completion of the detection measurement.
5. The method according to claim 1, provided with an electronic control system for controlling various cleaning procedures of the microfluidic system for the at least one viral detection chamber and rejuvenation procedures of the biosensors.
6. The method according to claim 1, utilizing an electric field which is perpendicular to the flow of liquid through the at least one viral detection chamber.
7. The method according to claim 1, wherein two to six viral detection chambers are operable in parallel to detect two to six aeropathogens simultaneously.
8. The method according to claim 1, wherein all fluids used are collected in a waste container.
9. The method according to claim 1, wherein the electronic processing system performs signal analysis and pattern recognition for the selected pathogens.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 shows an overall diagram of a preferred design of the device;
[0032] FIG. 2 shows a preferred design of the aerosampler;
[0033] FIGS. 3a and 3b show the preferred collector tubes in detail;
[0034] FIG. 4 shows the viral detection chamber;
[0035] FIG. 5 illustrates the construction of the viral detection chamber;
[0036] FIG. 6 shows the flow of the wetting fluid during continuous flow measurement;
[0037] FIG. 7 shows the cleaning procedure;
[0038] FIG. 8 shows schematically the binding mechanism of a virus to a gold electrode;
[0039] FIG. 9 shows a graph of a typical detection cycle as obtained by the present invention;
[0040] FIG. 10 shows examples of response of the system to different concentrations of a virus.
[0041] Equal numerals in different drawings designate the same functional elements.
DETAILED DESCRIPTION
[0042] With reference to FIG. 1, numeral 1 designates the aerosampler. A gas pump 13 sucks in air from the air inlet 11, preferably equipped with a dust collector 12. Within the aerosampler 1 small particle droplets entrained in the air are separated and collected as explained in in more detail with reference to FIGS. 2 and 3 below. Wetting liquid (buffer solution) is conveyed from container 2 by means of a micropump 21 into the aerosampler. The buffer solution with collected droplets is conveyed to the at least one viral detection chamber 4; shown are four viral detection chambers 4 in parallel, allowing for detecting four different aeropathogens simultaneously. To each of the viral detection chambers 4 is assigned an electronic detection system 5 with a common oscillator 6. The dashed lines represent electric connecting lines. Measured signals from the viral detection chamber 4 are electronically stored and processed in a computer 7. An assembly of liquid containers 3 containing liquids for cleaning, rinsing and refreshing may be included in the liquid circuit by three-way valve 31. These liquids may be conveyed by a micropump 91 (suction side of the pump) to the viral detection chamber 4 and after use to a waste container 9. Pump 91 also helps conveying the buffer solution from the aerosampler 1 to the viral detection chamber 4. Valve 92 is open when the detection scan is made with continuously flowing buffer solution through the viral detection chamber 4 and is closed when the detection scan is made batchwise, namely non-flowing buffer solution. All functions of the pumps, valves and electronic detection systems 5 are controlled via control lines 82 to controller (the embedded electronic system) 8 according to a preselected procedure. Preferably pump 92 provides a variable flow rate of from 15 nanoliters (nL) per minute to 15 μl per minute. Two pumps may be installed in parallel, one allowing high flow rates and the other low flow rates, which may be run alternatively.
[0043] With reference to FIG. 2, in a preferred embodiment air is conveyed by suction of pump 13 from air inlet 11 through micro-centrifuges 12, 15 and 16. Numeral 12 designates a durst filter. Preferably pump 13 is set at a flow rate of between 3.5 to 7.5 liters per minute (normal pressure). The dust outlet 17 may be equipped with a dust container 18 for collecting dust particles of diameter of above about 10 μm. The walls of the collector tubes 15 and 16 are wetted via line 22 from the wetting liquid container 2 (FIG. 1). Collector tube 15 preferably mainly collects particles with a diameter of from 2 μm to 10 μm, whereas collector tube 15 is adapted to collect particles mainly with diameter up to 5 μm. The wetting liquid having caught particles from the air collect on the bottom of the collector tubes 15 and 16 is conveyed via line 32 to the viral detection chamber 4. The collector tubes preferable have a total inner volume of between 1 ml and 2.5 ml.
[0044] FIG. 3a shows the airflow through the collector tubes 15 and 16. FIG. 3b shows collector tube 15 turned by 90° around its axis as compared to FIG. 3a. Air is injected in each case eccentrically and oblique to the bottom of the tube to create centrifugal force for separation of the particles entrained in the air.
[0045] FIG. 4 shows the viral detection chamber in detail. FIG. 4a) shows a top view, FIG. 4b) shows a side view and FIG. 4c) shows a front view. The designation of top, side or front view is only for defining the relative position of FIGS. 4 a), 4b) and 4c. The viral detection chamber may be arranged in any position, e.g. the front view may be the upper wall. The viral detection chamber, generally designated with 4, comprises an inner cavity 41 within a housing 401, 402 with an inlet 42 and an outlet 43. The arrows indicate the flow direction of the fluid received from the aerosampler 1. Preferably, the cavity 41 has a volume of between 0.5 and 1.5 mm.sup.3. A volume of between 0.6 to 1 mm.sup.3 is particularly preferred. The cavity 41 has parallel upper and lower walls carrying a number of counter electrodes 44 and working electrodes 45 opposite to each other. Preferably counter electrodes 44 are larger than the working electrodes that are carrying the binding agent for the aeropathogens. The smaller electrodes are the working electrodes. The distance between the working electrodes and the opposite counter electrode preferably is between 30 μm and 50 μm. The diameter of the counter electrodes 44 is preferably between 50 μm and up to 100 μm. The diameter of the working electrodes is preferably between 0.4 to 0.7 times the diameter of the counter electrodes. Each pair of working electrode and the opposite counter electrode create an electric field which is perpendicular to the flow direction of liquid. The distance of neighbouring counter electrodes is 15 to 30 times larger than their diameter. The electrodes are connected to connector terminals 47 and 48 as will be explained with reference to FIG. 5. An additional electrode 46 with connector terminals 49 serves as reference electrode. There is no working electrode opposite to the reference electrode. During detection the working electrodes are at a variable biasing voltage of −370 and +50 mV against the counter electrodes.
[0046] The housing of the viral detection is composed of two plates of preferably borosilicate float glass 401 and 402. Two holes 42 and 43 are drilled through one of the plates 402. Then the cavity is prepared by a reactive ion-CF.sub.4 plasma etch technique. It may be sufficient to provide a cavity in only one of the plates.
[0047] FIG. 5 a) and b) show the two plates 401 and 402 with their partial cavities on top. After providing the cavity 41, electrodes 44, 45 and 46, the connector terminals 47, 48 and 49 and the lines 441, 451 and 461 are applied to the plates by known photolithographic technique and deposition of noble metal vapour. The connecting lines 441, 451 and 461 are isolated by a suitable cover layer, e.g. of SiO.sub.2. After removal of any organic material from the surface of the plates, the plates are aligned to each other as indicated by the dotted line 403 in FIG. 4c). The exactly aligned plates 401 and 402 are then fused by heating to between about 500° C. and 650° C. under high pressure perpendicular to the fuse line 403.
[0048] FIG. 6 is a flow chart showing the flow of the wetting fluid from the moistening container to the waste container with flow rate controllers #1 and #2 cooperating with the controller 8.
[0049] FIG. 7 is a flow chart showing the cleaning/refreshing procedure. First (I) deionized water is conveyed through the device. Thereafter (II) cleaning liquid e.g. concentrated H.sub.2SO.sub.4 is flown through the viral detection chamber 4 from one of the containers 3, followed by rinsing with deionized water from another container 3. This may be repeated several times until constant current is measured from the electrodes of the viral detection chamber 4. Thereafter refreshing liquid is conveyed from another container 3. Refreshing liquid contains the functionalized aptamers (for a given aeropathogen) that will form the self-assembled monolayer on the working electrode.
[0050] FIG. 8 shows a typical binding mechanism for aeropathogens. First, the self-assembled monolayer on the working electrode is produced according known technique. In our initial experiments we used an amino- and thiol-modified aptamer having a methylene-blue reporter group in a 0.5 to about 1 μmolar aqueous buffer solution through the viral detection chamber. The formula of the self-assembled monolayer of aptamers was
5′-HS—(CH.sub.2).sub.6-GCAGT APTAMER ACTGCT-(CH.sub.2).sub.7—NH.sub.2-MB-3′.
or
5′-MB-NH.sub.2—(CH.sub.2).sub.7-TCGTCA-APTAMER-TGACG-(CH.sub.2).sub.6—HS-3′.
[0051] The left picture in FIG. 8 shows a single aptamer bound to a gold surface. The viral detection chamber is then rinsed with deionized water to remove the buffer solution.
[0052] Thereafter the liquid from the collector tubes is fed to the viral detection chamber. If aeropathogens are present in the liquid, what is indicated by the arrow and “virus” in the middle of FIG. 8, the aeropathogens binds to the aptamer by emitting an electron causing a response to the electronic detection system. After completion of the measurement, the viral detection chamber is rinsed with deionized water to remove all aeropathogens.
[0053] FIG. 9 shows a typical detection cycle, wherein the x-axis represents the bias-voltage and the y-axis represents the measured current. The upper solid curve obtained with no virus in the air is the calibration curve taken before each detection of ambient air. The lower dashed curve is obtained with viruses in the air. From the difference of areas below both curves, it is possible to estimate the concentration of virus in the air. Even if the aptamer is unspecific to the aeropathogen, information on the type of aeropathogen may be obtained from position of the maximum of the upper curve on the x-axis may. The upper dashed curve is obtained after removal of the aeropathogens of a previous detection for calibration for the next detection cycle.
[0054] FIG. 10 demonstrates preliminary experiments showing the electronic response of the system as a function of virus concentration. The detection chamber is first floated with a solution containing S1+H1N1-aptamers to cover the working electrodes with such aptamers. Then, deionized water is fed from one of the containers 3 to remove any buffer solution for 5 min (time 0-300 s of the diagram). Thereafter the biasing voltage is set to 235 mV for optimal binding and virus carrying air at a concentration of 200 nMol/l (represented as the concentration of Hemaggluttinin as measured by PCR=polymerase chain reaction)) is introduced into the aerosampler with continuous wetting the walls of the micro-centrifuges for another 5 min. The current drops to 7.5 nA, corresponding to a response of 1.8 nA. Then the supply of air is stopped, the biasing voltage is set to 100 mV and a BSL-solution (Phosphate Buffer Saline) with no viruses is injected from another container 3, whereby the viral particles are released from the sensors (“flush circle”) until the current returns to 9.3 nA. At the time of 900 s the BSL-solution is stopped and air with a virus concentration of 400 nMol/l is introduced into the aerosampler. The current now drops to 5.4 nA, corresponding to a response of 3.9 nA. Then this detection and flushing cycle is repeated. The last detection cycle is run with air containing 800 nMol/l viruses. The current drops to 0.8 nA, corresponding to a response of 8.5 nA. Accordingly, the response is nearly linear to the virus concentration in the air.
[0055] If after a number of detection/flushing cycles the current does not return to the original value (in the example 9.3 nA) the aptamers are removed from the detection chamber by flowing concentrated H.sub.2SO.sub.4 through the detection chamber with subsequent removing the acid with deionized water and renewing the aptamer coverage of the working electrodes.
[0056] In this preferred embodiment there are at least four containers 3 containing deionized water, BSL-solution, concentrated H.sub.2SO.sub.4 and a refreshing solution providing aptamers.
[0057] R&D resulting in this patent application has been funded in part by National Research Council of Canada (NRCC) and supported by NIOSH, Cincinnati, Ohio, USA, by providing the detailed drawings of the tandem reverse flow cyclones.
